US20020137906A1

US20020137906A1 - Tumor suppressor pathway in C. elegans

Info

Publication number: US20020137906A1
Application number: US09/872,523
Authority: US
Inventors: H. Horvitz; Ewa Davison; Xiaowei Lu
Original assignee: Individual
Current assignee: Massachusetts Institute of Technology
Priority date: 2000-06-02
Filing date: 2001-06-01
Publication date: 2002-09-26
Also published as: WO2001094545A3; US20040229267A1; US7670778B2; WO2001094545A2; US20080213777A1; AU2001275163A1

Abstract

The invention provides novel lin-8, lin-56, and lin-61 genes and polypeptides involved in cell fate determination and in cell proliferation. In addition, the invention includes mutants of these three genes, as well as methods for utilizing these genes, and their encoded polypeptides, in diagnosing and treating abnormal cell proliferation.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. provisional application No. 60/208,802, filed on Jun. 2, 2000, which is hereby incorporated by reference.[0001]

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] The present research was supported by a grant from the National Institutes of Health (Number GM 24663). The U.S. government has certain rights to this invention.

BACKGROUND OF THE INVENTION

The field of the invention is cell proliferation.

The nematode Caenorhabditis elegans (C. elegans) is well suited for developmental genetic studies because the entire cell lineage has been mapped and is essentially invariant from one animal to the next. Thus, by comparing the cell lineage of a wild-type animal to that of a mutant animal, the changes in cell fates caused by the mutation can be determined.

A number of mutations that alter cell lineage in C. elegans, termed lin mutations, were obtained in genetic screens conducted by Horvitz and Sulston in the late 1970's. A subset of the mutations affected the formation of the vulva, a structure on the ventral surface of C. elegans hermaphrodites through which eggs are laid and through which sperm enters during cross-fertilization. Six vulval precursor cells have the potential to undertake a vulval cell lineage, as defmed by the number and pattern of cell divisions. In a wild-type animal only three of these cells actually undertake vulval cell fates and these three cells generate the 22 cells that make up the adult vulva. In multivulva (Muv) animals, most or all of the six vulval precursor cells undertake vulval cell fates. In addition to the cells required for the formation of a normal vulva, these mutant animals generate an excess of cells which cause the formation of raised, vulva-like structures on the ventral surface of the animal. On the other hand, a vulvaless (Vul) phenotype results when no or too few vulval precursor cells adopt vulval cell fates.

Genetic and molecular analyses of Muv and Vul animals have defined a Ras signal transduction pathway that mediates induction of the hermaphrodite vulva. This pathway includes the LIN-3 EGF-like ligand, the LET-23 receptor tyrosine kinase, the SEM-5 adaptor, LET-60 Ras, the KSR-1 kinase, LIN-45 Raf, MEK-2, and the MPK-1 MAP kinase, and regulates the activities of the ETS transcription factor LIN-1 and the winged-helix transcription factor LIN-31 (reviewed by Horvitz and Sternberg, Nature 351:535-41, 1991; Sundaram and Han, Bioessays 18:473-480, 1996; Tan et al., Cell 93:569-580, 1998). Mutant animals in which this pathway is ectopically activated can display a Muv phenotype, whereas mutant animals that have reduced Ras pathway signaling can display a Vul phenotype.

The synthetic multivulva (synMuv) genes act in two functionally-redundant pathways as negative regulators of the nematode Ras signaling pathway. The first synthetic multivulva mutant was identified by Horvitz and Sulston. The two genetic loci mutated in this mutant were termed lin-8 and lin-9. Reduction-of-function mutations in both of these loci were required for a multivulva phenotype. Subsequent genetic screens identified a set of loci which fall into the same class as lin-8, termed class A genes, and genes which fall into the same class as lin-9, termed class B genes. In general, an animal with a reduction-of-function mutation in any class A gene and a reduction-of-function mutation in any class B gene will display a multivulva phenotype, while animals carrying one or more mutations of the same class have a wild-type vulval phenotype. These two classes appear to define two functionally redundant pathways that negatively regulate the expression of vulval cell fates.

Thus far at least four class A loci (lin-8, lin-15A, lin-38, and lin-56) and at least fourteen class B loci (lin-9, lin-15B, lin-35, lin-36, lin-37, lin-13, lin-52, lin-53, lin-5 lin-55 (dpl-1), lin-61, hda-1, tam-1 (Hsieh et al., Genes & Dev. 13:2958-2970, 1999), and the C. elegans E2F1 homolog (efl-1)) have been identified genetically. lin-15 encodes both A and B activities in two non-overlapping transcripts. In addition, lin-37, lin-35, lin-53, lin-52, lin-54, lin-55 (dpl-1), lin-15A, lin-15B, lin-36, lin-9, lin-55, and efl-1 have been cloned (Ceol and Horvitz, Molecular Cell 7:461-473, 2001; Clark et al., Genetics 137:987-997, 1994; Huang et al., Mol. Biol. Cell 5:395-411, 1994; Beitel et al., Gene 254:253-263, 2000; and PCT WO 98/54299).

A number of the synMuv family members encode polypeptides with sequence similarity to polypeptides involved in cancer development and progression. For example, lin-35 encodes a homolog of the mammalian pocket protein family, which includes retinoblastoma protein (Rb), p107, and p130. This family of proteins has been the subject of intense study since the cloning of Rb in 1986. Rb is a tumor suppressor gene; mutations that inactivate Rb predispose individuals to tumor formation. Most commonly, inactivation of Rb results in a type of eye cancer, retinoblastoma, although inactivating mutations in Rb have been found in other types of tumors. The Rb protein is thought to function as a negative regulator of cell cycle progression. A number of molecules that interact, both directly and indirectly, with Rb and the other pocket proteins have been characterized in mammalian cells.

Another synMuv family member, lin-53, encodes a homolog of p48, a protein which has been shown to bind Rb. Although the functional significance of the interaction between p48 and Rb is not fully understood, recent studies suggest that p48 may play a role in remodeling chromatin structure. In addition, lin-55(dpl-1) encodes a homolog of the DP family of proteins (Ceol and Horvitz, Molecular Cell 7:461-473, 2001). DP family members, together with E2F proteins, bind DNA at specific sites, thereby regulating the transcription of genes that are essential for cell cycle progression. Furthermore, pocket proteins such as Rb bind to the DP-E2F complex to repress transcription.

As in the nematode, Ras pathways have been found to control cell proliferation in a range of organisms from the yeast Saccharomyces cerevisiae to humans. The Ras pathway defines one class of oncogene signaling pathways; members of this pathway, most commonly Ras itself, have been shown to be mutated in a broad range of human cancers (Hunter, Cell 88:333-346, 1997). Accordingly, analysis of the Ras pathway, in particular the vulval induction pathway, in C. elegans addresses the significant need of increasing our understanding of cancer in general.

SUMMARY OF THE INVENTION

We isolated and cloned three novel C. elegans genes, lin-8, lin-56, and lin-61, that are part of two synMuv pathways and we characterized several mutations in these genes. lin-8, lin-56, and lin 61 genes, their mutants, and the proteins they encode, may be used in genetic and biochemical model systems to further our understanding of cell proliferative diseases, including cancer, as well as in diagnosing and treating cell proliferative diseases.

Accordingly, the first aspect of the invention features a substantially pure nucleic acid encoding a LIN-8 polypeptide, where the LIN-8 polypeptide includes at least 130 contiguous amino acids of SEQ ID NO:1 and modulates cell proliferation. In a preferred embodiment of this aspect of the invention, the amino acid sequence of the LIN-8 polypeptide includes SEQ ID NO:1, and in another embodiment, the LIN-8 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:1, for example, one that increases cell proliferation.

In other preferred embodiments of this aspect, the polynucleotide sequence of the nucleic acid includes SEQ ID NO:2, or at least 400 contiguous nucleotides of SEQ ID NO:2. In addition, the polynucleotide sequence of the nucleic acid may include a mutant lin-8 nucleic acid sequence, for example, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.

A second aspect of the invention features a polypeptide having an amino acid sequence identical to any one of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47.

In a third aspect, the invention encompasses a substantially pure nucleic acid encoding a LIN-56 polypeptide, where the LIN-56 polypeptide includes at least 110 contiguous amino acids of SEQ ID NO:3 and modulates cell proliferation. In addition, the amino acid sequence of the LIN-56 polypeptide may be SEQ ID NO:3. In a preferred embodiment of this aspect of the invention, the LIN-56 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:3, for example, one that increases cell proliferation.

In additional embodiments of this aspect of the invention, the polynucleotide sequence of the nucleic acid includes SEQ ID NO:4, or at least 400 contiguous nucleotides of SEQ ID NO:4. Furthermore, the polynucleotide sequence of the nucleic acid may be a mutant lin-56 nucleic acid sequence, such as SEQ ID NO:48.

A fourth aspect of the invention features a substantially pure nucleic acid encoding a LIN-61 polypeptide, where the LIN-61 polypeptide includes at least 130 contiguous amino acids of SEQ ID NO:5 and modulates cell proliferation. In preferred embodiments of this aspect, the amino acid sequence of the LIN-61 polypeptide may be SEQ ID NO:5, or the LIN-61 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:5, for example, one that increases cell proliferation.

In another embodiment of this aspect of the invention, the polynucleotide sequence of the nucleic acid may be SEQ ID NO:6, or it may include at least 400 contiguous nucleotides of SEQ ID NO:6. Furthermore, the polynucleotide sequence of the nucleic acid may be a mutant lin-61 nucleic acid sequence, for example, one having the sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:78.

A fifth aspect of the invention encompasses a polypeptide having an amino acid sequence identical to any one of SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.

In a sixth aspect, the invention features a vector including a nucleic acid having a polynucleotide sequence, for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. Preferably, this vector is capable of directing expression of the nucleic acid, for example, in a cell.

A seventh aspect of the invention encompasses a transgenic cell including a nucleic acid sequence encoding a lin-8, a lin-56, or a lin-61 polypeptide, where the nucleic acid sequence is located in the genome of the cell in a position in which it does not naturally occur. In addition, the nucleic acid sequence may be operably linked to a heterologous promoter.

Additional aspects of the invention feature purified antibodies which specifically bind to a LIN-8, LIN-56, or LIN-61 polypeptide.

In further aspects, the invention provides methods of modulating proliferation of a cell which involve administering a proliferation-modulating amount of a polypeptide, or a nucleic acid encoding a polypeptide, having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 to a cell, for example, a mammalian cell, such as a human cell. In addition, the nucleic acid sequence may be contained in a vector.

In another aspect, the invention features a method of identifying a compound that modulates cell proliferation, involving: (a) providing a cell expressing a nucleic acid, for example, a lin-8, lin-56, or lin-61 nucleic acid, or a reporter gene, operably linked to a lin-8, lin-56, or lin-61 promoter; (b) contacting the cell with a candidate compound; and (c) measuring the expression of the nucleic acid, where an alteration in the level of expression of the nucleic acid indicates the presence of a compound that modulates cell proliferation. In preferred embodiments, step (c) involves measuring the expression of the protein encoded by the nucleic acid, for example by using an antibody that specifically binds to a LIN-8, LIN-56, or LIN-61 polypeptide, or step (c) may also involve measuring the mRNA level of the nucleic acid.

An additional aspect of the invention provides a method of identifying a candidate compound, for example, a polypeptide, that binds to a LIN-8, LIN-56, or LIN-61 polypeptide, involving: (a) providing the polypeptide; (b) contacting the polypeptide with a candidate compound; and (c) measuring the binding of the candidate compound to the polypeptide, where the binding indicates the presence of a candidate compound that binds a LIN-8, LIN-56, or LIN-61 polypeptide.

Furthermore, in another aspect, the invention provides a method of diagnosing an animal, for example, a mammal, such as a human, for the presence of a cell proliferation disease, such as cancer, or for an increased likelihood of developing a cell proliferation disease. This method involves determining whether a nucleic acid sample obtained from the animal includes a mutant lin-8, lin-56, or lin-61 nucleic acid, where the presence of the mutant lin-8, lin-56, or lin-61 nucleic acid indicates that the animal has a cell proliferation disease, or is at an increased likelihood of developing a cell proliferation disease. In preferred embodiments of this aspect of the invention, the mutant lin-8 nucleic acid may be, for example, lin-8(n2738), lin-8(n2731), lin-8(n3606), lin-8(n3595), lin-8(n2739), lin-8(n3586), lin-8(n3588), lin-8(n-111), lin-8(n2741), lin-8(n3585), lin-8(n3646), lin-8(n2376), lin-8(n2378), lin-8(n2403), lin-8(n2724), lin-8(n3607), lin-8(n3591), lin-8(n3609), or lin-8(n3581), the mutant lin-56 nucleic acid may be, for example, lin-56(n3355) or lin-56(n2728), and the mutant lin-61 nucleic acid may be, for example, lin-61(n3446), lin-61(n3447), lin-61 (n3624), or lin-61(n3635).

In an additional aspect, the invention features a method of diagnosing an animal, for example, a mammal, such as a human, for the presence of a cell proliferation disease, or an increased likelihood of developing a cell proliferation disease. This method involves measuring lin-8, lin-56, or lin-61 nucleic acid expression in a sample obtained from the animal, where an alteration in the expression, relative to a sample obtained from an unaffected animal, indicates that the animal has a cell proliferation disease, or an increased likelihood of developing a cell proliferation disease. In a preferred embodiment of this aspect, nucleic acid expression is measured by measuring the amount of the LIN-8, LIN-56, or LIN-61 polypeptide, for example, by using an antibody that specifically binds to a LIN-8, LIN-56, or LIN-61 polypeptide in the sample. However, nucleic acid expression may also be measured by measuring the amount of lin-8, lin-56, or lin-61 mRNA in the sample.

In a final aspect, the invention provides a method of identifying a nucleic acid that modulates cell proliferation. This method involves: (a) expressing in a cell (i) a first nucleic acid operably linked to a first promoter, where the first promoter may be the lin-8, lin-56, or lin-61 promoter; and (ii) a second nucleic acid operably linked to a second promoter; and (b) measuring the expression of the first nucleic acid, where a modulation in the expression of the first nucleic acid in the presence of the second nucleic acid, indicates that the second nucleic acid modulates cell proliferation. In a preferred embodiment of this aspect, the first nucleic acid is a lin-8, lin-56, or lin-61 nucleic acid. Furthermore, the measuring in step (b) may also involve comparing the amount of modulation in the expression of the first nucleic acid seen in the presence of the second nucleic acid with that seen in the presence of a control nucleic acid that does not modulate cell proliferation.

Definitions

By a “lin-8 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, that is substantially identical to SEQ ID NO:2, or portions thereof. Preferably a “lin-8 nucleic acid” is identical to at least 100, 200, 300, 390, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, or 1200 contiguous nucleotides of SEQ ID NO:2, its complement, or to the corresponding RNA sequence. However, a “lin-8 nucleic acid” may also be identical to SEQ ID NO:2. In addition, a “lin-8 nucleic acid” may be characterized by its ability to modulate cell proliferation. Furthermore, a “lin-8 nucleic acid” may refer to a nucleic acid sequence including nucleic acids 375-989, 400-900, 450-850, 500-800, or 550-750 of SEQ ID NO:2, or to a nucleic acid that hybridizes under highly stringent conditions to these regions of SEQ ID NO:2. For example, highly stringent conditions include hybridization at about 42° C. and about 50% formamide, 0.1 mg/nl sheared salmon sperm DNA, 1% SDS, 2×SSC, 10% Dextran sulfate, a first wash at about 65° C., about 2×SSC, and 1% SDS, followed by a second wash at about 65° C. and about O.1×SSC. Alternatively, highly stringent conditions may include hybridization at about 42° C. and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE, 1×Denhardt's, followed by two washes at room temperature and 2×SSC, 0.1% SDS, and two washes at between 55-60° C. and 0.2×SSC, 0.1% SDS. The terms “gene” and “nucleic acid sequence” may be used interchangeably.

By a “mutant lin-8 nucleic acid” or a “mutated lin-8 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, differing from the wild-type lin-8 nucleic acid sequence by at least one nucleotide. This nucleotide difference may result, for example, in the “mutant lin-8 nucleic acid” encoding a truncated LIN-8 protein or one containing a missense mutation. Preferably, the “mutant lin-8 nucleic acid” is identical to at least 100, 200, 300, 390, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ ID NO:2, its complement, or the corresponding mRNA sequence. Most preferably, the “mutant lin-8 nucleic acid” is the lin-8(n2738), lin-8(n2731), lin-8(n3606), lin-8(n3595) lin-8(n2739), lin-8(n3586), lin-8(n3588), lin-8(n-111), lin-8(n2741), lin-8(n3585), lin-8(n3646), lin-8(n2376), lin-8(n2378), lin-8(n2403), lin-8(n2724), lin-8(n3607), lin-8(n3591), lin-8(n3609), or lin-8(n3581) nucleic acid sequence. Furthermore, a C. elegans carrying a mutant lin-8 nucleic acid sequence and a reduction of function mutation in any synMuv class B gene will display a multivulva phenotype.

By a “lin-56 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, that is substantially identical to SEQ ID NO:4, or portions thereof. Preferably a “lin-56 nucleic acid” is identical to at least 100, 200, 300, 330, 390, 400, 450, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ ID NO:4, its complement, or to the corresponding RNA sequence. However, a “lin-56 nucleic acid” may also be identical to SEQ ID NO:4. In addition, a “lin-56 nucleic acid” may be characterized by its ability to modulate cell proliferation. Furthermore, a “lin-56 nucleic acid” may refer to a nucleic acid sequence including nucleic acids 376-1108, 400-1000, 400-700, 450-950, 450-800, 500-900, 550-850, or 600-800 of SEQ ID NO:4, or to a nucleic acid that hybridizes under highly stringent conditions to these regions of SEQ ID NO:4. For example, highly stringent conditions include hybridization at about 42° C. and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 1% SDS, 2×SSC, 10% Dextran sulfate, a first wash at about 65° C., about 2×SSC, and 1% SDS, followed by a second wash at about 65° C. and about0.1×SSC. Alternatively, highly stringent conditions may include hybridization at about 42° C. and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE, 1×Denhardt's, followed by two washes at room temperature and 2×SSC, 0.1% SDS, and two washes at between 55-60° C. and 0.2×SSC, 0.1% SDS.

By a “mutant lin-56 nucleic acid” or a “mutated lin-56 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, differing from the wild-type lin-56 nucleic acid sequence by at least one nucleotide. This nucleotide difference may result, for example, in the “mutant lin-56 nucleic acid” encoding a truncated LIN-56 protein or one containing a missense mutation. Preferably, the “mutant lin-56 nucleic acid sequence” is identical to at least 100, 200, 330, 390, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ ID NO:4, its complement, or the corresponding mRNA sequence. Most preferably, the “mutant lin-56 nucleic acid” is the lin-56(n3355) nucleic acid sequence. Furthermore, a C. elegans carrying a mutant lin-56 nucleic acid sequence and a reduction of function mutation in any synMuv class B gene will display a multivulva phenotype.

By a “lin-61 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, that is substantially identical to SEQ ID NO:6, or portions thereof. Preferably a “lin-61 nucleic acid” is identical to at least 100, 200, 300, 390, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 contiguous nucleotides of SEQ ID NO:6, its complement, or to the corresponding RNA sequence. However, a “lin-61 nucleic acid” may also be identical to SEQ ID NO:6. In addition, a “lin-61 nucleic acid” may be characterized by its ability to modulate cell proliferation. Furthermore, a “lin-61 nucleic acid” may refer to a nucleic acid sequence including nucleic acids 375-1150, 400-1100, 400-1000, 450-950, 500-1000, 500-900, or 550-850 of SEQ ID NO:6, or to a nucleic acid that hybridizes under highly stringent conditions to these regions of SEQ ID NO:6. For example, highly stringent conditions include hybridization at about 42° C. and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 1% SDS, 2×SSC, 10% Dextran sulfate, a first wash at about 65° C., about 2×SSC, and 1% SDS, followed by a second wash at about 65° C. and about 0,1×SSC. Alternatively, highly stringent conditions may include hybridization at about 42° C. and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5×SSPE, 1×Denhardt's, followed by two washes at room temperature and 2×SSC, 0.1% SDS, and two washes at between 55-60° C. and 0.2×SSC, 0.1% SDS.

However, a “lin-61 nucleic acid” may also be identical to at least 100, 200, 300, 390, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 contiguous nucleotides of SEQ ID NO:76, its complement, or to the corresponding RNA sequence. In addition, a “lin-61 nucleic acid” may be identical to SEQ ID NO:76.

By a “mutant lin-61 nucleic acid” or a “mutated lin-61 nucleic acid” is meant a nucleic acid sequence, or fragment thereof, differing from the wild-type lin-61 nucleic acid sequence by at least one nucleotide. This nucleotide difference may result, for example, in the “mutant lin-61 nucleic acid” encoding a truncated LIN-61 protein or one containing a missense mutation. Preferably, the “mutant lin-61 nucleic acid” is identical to at least 100, 200, 300, 390, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 contiguous nucleotides of SEQ ID NO:6, its complement, or the corresponding mRNA sequence. Most preferably, the “mutant lin-61 nucleic acid” is the lin-61(n3446), lin-61(n3447),or lin-61(n3624) nucleic acid sequence. However, a mutant “lin-61 nucleic acid,” for example, a lin-61(sy223) or lin-61(n3635) nucleic acid sequence, may also be identical to at least 100, 200, 300, 390, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 contiguous nucleotides of SEQ ID NO:76, its complement, or the corresponding mRNA sequence. Furthermore, a C. elegans carrying a mutant lin-61 nucleic acid sequence and a reduction of function mutation in any synMuv class A gene will display a multivulva phenotype.

By a “lin-8(n111) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:16, its complement, or the corresponding RNA sequence.

By a “lin-8(n2741) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:18, its complement, or the corresponding RNA sequence.

By a “lin-8(n2738) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:20, its complement, or the corresponding RNA sequence.

By a “lin-8(n2731) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:22, its complement, or the corresponding RNA sequence.

By a “lin-8(n3585) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:24, its complement, or the corresponding RNA sequence.

By a “lin-8(n3646) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:26, its complement, or the corresponding RNA sequence.

By a “lin-8(n3606) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:28, its complement, or the corresponding RNA sequence.

By a “lin-8(n23 76) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:30, its complement, or the corresponding RNA sequence.

By a “lin-8(n2378) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:32, its complement, or the corresponding RNA sequence.

By a “lin-8(n3595) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:34, its complement, or the corresponding RNA sequence.

By a “lin-8(n2403) nucleic acid,” a “lin-8(2724) nucleic acid,” or a “lin-8(3607) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:36, its complement, or the corresponding RNA sequence.

By a “lin-8(n3581) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:38, its complement, or the corresponding RNA sequence.

By a “lin-8(n3609) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:40, its complement, or the corresponding RNA sequence.

By a “lin-8(n2739) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:42, its complement, or the corresponding RNA sequence.

By a “lin-8(n3586) nucleic acid,” or a “lin-8(n3588) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:44, its complement, or the corresponding RNA sequence.

By a “lin-8(n3591) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:46, its complement, or the corresponding RNA sequence.

By a “lin-56(n3355) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:48, its complement, or the corresponding RNA sequence.

By a “lin-56(n2728) nucleic acid” is meant a deletion encompassing all or part of a lin-56 nucleic acid sequence in an isolated nucleic acid containing the genomic region that normally contains a lin-56 nucleic acid sequence.

By a “lin-61(n3446) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:73, its complement, or the corresponding RNA sequence.

By a “lin-61(n3447) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:74, its complement, or the corresponding RNA sequence.

By a “lin-61(n3624) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:75, its complement, or the corresponding RNA sequence.

By a “lin-61(sy223) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:77, its complement, or the corresponding RNA sequence.

By a “lin-61(n3635) nucleic acid” is meant a nucleic acid sequence having SEQ ID NO:78, its complement, or the corresponding RNA sequence.

By “LIN-8 polypeptide” or “LIN-8 protein” is meant a polypeptide or protein encoded by a lin-8 nucleic acid sequence. Preferably, a “LIN-8 polypeptide” is identical to at least 30, 50, 100, 130, 200, 250, 300, or 350 contiguous amino acids of SEQ ID NO:1. Most preferably, a “LIN-8 polypeptide” is identical to SEQ ID NO:1 and has a LIN-8 biological activity described below.

By a “mutant LIN-8 polypeptide” is meant a LIN-8 polypeptide that differs by at least one amino acid from a wild-type LIN-8 polypeptide. In addition, a mutant LIN-8 polypeptide may also be a truncated protein, for example, due to the presence of a premature stop codon in the nucleic acid sequence encoding the mutant LIN-8 polypeptide. In addition, a “mutant LIN-8 polypeptide” is preferably 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identical to at least 30, 50, 100, 130, 200, 250, 300, or 350 contiguous amino acids of SEQ ID NO:1. Furthermore, the mutant LIN-8 polypeptide preferably is the polypeptide encoded by lin-8(n2738), lin-8(n2731), lin-8(n3606), lin-8(n3595), lin-8(n2739), lin-8(n3586), lin-8(n3588), lin-8(n-111), lin-8(n2741), lin-8(n3585), lin-8(n3646), lin-8(n2376), lin-8(n2378), lin-8(n2403), lin-8(n2724), lin-8(n3607), lin-8(n3591), lin-8(n3609), or lin-8(n3581). Most preferably “mutant LIN-8 polypeptide” is identical to SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47.

By “LIN-56 polypeptide” or “LIN-56 protein” is meant a polypeptide or protein encoded by a lin-56 nucleic acid sequence. Preferably, a “LIN-56 polypeptide” is identical to at least 30, 50, 100, 130, 200, 250, or 300 contiguous amino acids of SEQ ID NO:3. Most preferably, a “LIN-56 polypeptide” is identical to SEQ ID NO:3 and has a LIN-56 biological activity described below.

By a “mutant LIN-56 polypeptide” is meant a LIN-56 polypeptide that differs by at least one amino acid from a wild-type LIN-56 polypeptide. In addition, a mutant LIN-56 polypeptide may also be a truncated protein, for example, due to the presence of a premature stop codon in the nucleic acid sequence encoding the mutant LIN-56 polypeptide. In addition, a “mutant LIN-56 polypeptide” is preferably 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identical to at least 30, 50, 100, 130, 200, 250, or 300 contiguous amino acids SEQ ID NO:3. Furthermore, the mutant LIN-56 polypeptide is preferably the polypeptide encoded by a lin-56(n3355) nucleic acid.

By “LIN-61 polypeptide” or “LIN-61 protein” is meant a polypeptide or protein encoded by a lin-61 nucleic acid sequence. Preferably, a “LIN-61 polypeptide” is identical to at least 30, 50, 100, 130, 200, 250, 300, 350, or 400 contiguous amino acids of SEQ ID NO:5. Most preferably, a “LIN-61 polypeptide” is identical to SEQ ID NO:5 and has a LIN-61 biological activity described below.

By a “mutant LIN-61 polypeptide” is meant a LIN-61 polypeptide that differs by at least one amino acid from a wild-type LIN-61 polypeptide. In addition, a mutant LIN-61 polypeptide may also be a truncated protein, for example, due to the presence of a premature stop codon in the nucleic acid sequence encoding the mutant LIN-61 polypeptide. In addition, a “mutant LIN-61 polypeptide” is preferably 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identical to at least 30, 50, 100, 130, 200, 250, 300, 350, or 400 contiguous amino acids SEQ ID NO:5. Preferably, a “mutant LIN-61 polypeptide” is encoded by a lin-61(n3446), lin-61(n3447), or lin-61(3624) nucleic acid. Most preferably, a “mutant LIN-61 polypeptide” is identical to SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.

By an “amino acid alteration,” as used herein, is meant a change in an amino acid sequence, relative to the wild-type sequence. Such a change may be, for example, the substitution of one or more amino acids with heterologous amino acids, as well as the addition or deletion of one or more amino acids. In addition, an “amino acid alteration” may result in a truncated protein.

By “heterologous promoter” is meant a nucleic acid sequence that drives expression of a nucleic acid sequence, e.g., a gene, with which is not naturally associated.

By a “synMuv gene” is meant a nucleic acid sequence encoding LIN-9, LIN-15A, LIN-15B, LIN-37, LIN-35, LIN-53, LIN-55, LIN-52, LIN-54, and the E2F-1 gene of C. elegans, and the LIN-54 genes of the mouse and human. SynMuv genes also include those which encode polypeptides encoded by ESTs zp44h06.sl, zr79e11.r1, and EST180962 and any other nucleic acid sequence identified as a synMuv sequence known in the art.

By “synMuv polypeptide” or “synMuv protein” is meant a polypeptide encoded by a synMuv gene.

By “LIN-8 biological activity,” “LIN-56 biological activity,” or “LIN-61 biological activity” is meant an activity of a LIN-8, LIN-56, or LIN-61 polypeptide, when expressed or overexpressed, either alone or in combination, with another polypeptide in a cell, which is absent or decreased in the absence of the LIN-8, LIN-56, or LIN-61 polypeptide. A LIN-8, LIN-56, or LIN-61 biological activity includes modulating or altering cell proliferation. Another activity is rescuing (i.e., suppressing) a LIN-8, LIN-56, or LIN-61 mutant phenotype. Less preferably, a LIN-8, LIN-56, or LIN-61 biological activity involves binding to other known synMuv polypeptides, in vivo or in vitro. Another LIN-8, LIN-56, or LIN-61 biological activity is binding to an antibody that recognizes a LIN-8, LIN-56, or LIN-61 polypeptide. Finally, a LIN-8, LIN-56, or LIN-61 biological activity may also be the ability of the nucleic acid sequence encoding the polypeptide to hybridize to a detectably-labeled probe from a lin-8, lin-56, or lin-61 nucleic acid sequence under high, or less preferably, low stringency conditions.

By “modulating cell proliferation” or “altering cell proliferation” is meant increasing or decreasing the number of cells which undergo cell division in a given cell population or altering the fate of a given cell. It will be appreciated that the degree of modulation provided by LIN-8, LIN-56, LIN-61, or a modulatory compound, in a given assay will vary, but that one skilled in the art can determine the statistically significant change (e.g., a p-value≦0.05) in the level of cell proliferation which identifies a modulatory compound.

By “inhibiting cell proliferation” is meant any decrease in the number of cells that undergo division relative to an untreated control. Preferably, the decrease is at least 25%, more preferably the decrease is at least 50%, and most preferably the decrease is at least 75%, 80%, or even 100%.

By “a cell proliferation disease,” is meant a disorder that is due to any genetic alteration within a differentiated cell that results in the abnormal proliferation of the cell. Examples of such changes include mutations in genes involved in the regulation of the cell cycle, of growth control, or of apoptosis, and further can include tumor suppressor genes and proto-oncogenes. In addition, “a cell proliferation disease” may be the result of, for example, an inappropriately high level of cell division, or an inappropriately low level of apoptosis, or both. Specific examples of cell proliferation diseases are various types of cancer including cancers of the reproductive system, such as cervical cancer and ovarian cancer.

By “unaffected animal,” as used herein, is meant an animal that does not have, or is not at an increased likelihood of developing, a cell proliferation disease.

By “specifically binds,” as used herein in reference to an antibody, is meant an antibody that recognizes a specific protein, or shows staining in a sample, but does not recognize a specific protein, or show staining, in a sample not containing the protein of interest. Assays used to determine binding include, for example, Western blotting, affinity column purification, and tissue staining. A sample not containing the protein of interest may be obtained from an organism mutant for the protein of interest.

By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 60%, 70%, 75%, or 80%, more preferably 90% or 95%, and most preferably 99% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25, 35, 50, 75, 100, 110, 130, 150, 200, 250, 300, or 310 amino acids, and most preferably the full-length amino acid sequence. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75, 110, 200, 330, 390, 450, 600, 800, 900, or 1000 nucleotides, and most preferably the full-length nucleotide sequence.

Sequence identity may be measured using sequence analysis software on the default setting (i.e., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

A polypeptide which is substantially identical to a LIN-8, LIN-56, or LIN-61 polypeptide (SEQ ID NOS: 1, 3, or 5) may be, for example, another substantially pure naturally-occurring mammalian LIN-8, LIN-56, or LIN-61 polypeptide as well as an allelic variant; a natural mutant; an induced mutant; and a DNA sequence that encodes a polypeptide. In addition, such polypeptides may also be any polypeptides specifically bound by antisera directed to a LIN-8, LIN-56, or LIN-61 polypeptide. Furthermore, polypeptides that are substantially identical to LIN-8, LIN-56, or LIN-61 polypeptides also include chimeric polypeptides that have a LIN-8, LIN-56, or LIN-61 polypeptide portion. Preferably, this LIN-8, LIN-56, or LIN-61 polypeptide portion contains at least 50, 75, 90, 110, 130, 150, 200, 250, or 300 contiguous amino acids of SEQ ID NOS:1, 3, or5.

By a “substantially pure polypeptide” is meant a polypeptide, for example, LIN-8, LIN-56, or LIN-61, that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a LIN-8, LIN-56, or LIN-61 polypeptide. A substantially pure LIN-8, LIN-56, or LIN-61 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte cell); by expression of a recombinant nucleic acid encoding a LIN-8, LIN-56, or LIN-61 polypeptide; or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants, which accompany it in its natural state. Thus, a protein, which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates, will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.

By a “substantially pure DNA” or “a substantially pure nucleic acid sequence” is meant a nucleic acid sequence or DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid sequence or DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence or an antisense DNA or RNA sequence.

By “antisense” is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand gene encoding a nucleic acid sequence of interest, for example, a lin-8, lin-56, or lin-61 nucleic acid sequence. Preferably, the antisense nucleic acid is capable of decreasing the biological activity of the polypeptide encoded by the nucleic acid sequence of interest when present in a cell. Preferably the decrease is at least 10%, relative to a control, more preferably 25%, 50%, or 75%, and most preferably 100%.

By “analog of LIN-8”, “analog of LIN-56”, or “analog of LIN-61,” is meant differing from a naturally-occurring LIN-8, LIN-56, or LIN-61 polypeptide by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring LIN-8, LIN-56, or LIN-61 amino acid sequence (SEQ ID NOS:1, 3, or 5). The length of sequence comparison is at least 25, 50, 75, 100, 110, 130, 150, 200, 250, or 300 contiguous amino acid residues, and more preferably more than 310 amino acid residues, for example, the full-length sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.

In addition, analogs may differ from the naturally-occurring LIN-8, LIN-56, or LIN-61 polypeptide by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate (EMS) or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), Cold Spring Harbor Press, 1989, or Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 2000). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally-occurring or synthetic amino acids, e.g.,β or γ amino acids.

As used herein, the term “LIN-8 polypeptide fragment”, “LIN-56 polypeptide fragment”, or “LIN-61 polypeptide fragment,” means at least 20 contiguous amino acids, preferably at least 50 contiguous amino acids, more preferably at least 100 contiguous amino acids, and most preferably at least 110, 130, 150, 200, 250, 300, 310 or more contiguous amino acids of SEQ ID NOS:1, 3, or 5. Fragments of LIN-8, LIN-56, or LIN-61 polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). Preferable fragments or analogs according to the invention are those which facilitate specific detection of a lin-8, lin-56, or lin-61 nucleic acid or amino acid sequence in a sample to be diagnosed.

By “transformed cell” is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a LIN-8, LIN-56, LIN-61 polypeptide, or a reporter gene.

By “transgene” is meant any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from that cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. Self-replicating units, such as artificial chromosomes, are included.

By “transgenic” is meant any cell that includes a DNA sequence inserted by artifice into a cell and that becomes part of the genome of the organism that develops from that cell. As used herein, the transgenic organism is generally a transgenic nematode (e.g., C. elegans), non-human mammal (e.g., a rodent such as a rat or mouse), or a plant, and the DNA sequence (transgene) is inserted by artifice into the nuclear genome.

By “transformation” is meant any method for introducing foreign molecules into a cell. Microinjection, lipofection, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the teachings which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts, which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles, bacteria, yeast, fungi, algae, animal tissue, and cultured cells.

By “positioned for expression” is meant that a nucleic acid molecule is positioned adjacent to a nucleic acid sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a LIN-8, LIN-56, or LIN-61 polypeptide, a recombinant protein or an RNA molecule).

By “reporter gene” is meant a gene whose expression may be assayed; such genes include, without limitation, those encoding glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and β-galactosidase.

By “promoter” is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements that are sufficient to render promoter-dependent gene expression controllable for specific cell-types or tissues. In addition, such promoters may also render gene expression inducible by external signals or agents. These promoter elements may be located in the 5′ or 3′ regions of the native gene, for example, a lin-8, lin-56, or lin-61 gene.

By “operably linked” is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

By “detectably-labeled” is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as ³²p or ³⁵S) and nonradioactive labeling (e.g., chemiluminescent labeling, such as fluorescein labeling).

By “purified antibody” is meant an antibody that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 80%, 85%, or 90%, and most preferably at least 99%, by weight, antibody, e.g., a LIN-8, LIN-56, or LIN-61 specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.

By “specifically binds” is meant an antibody which recognizes and binds a protein, but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes additional proteins.

By a “candidate compound” or “test compound” is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate cell proliferation, by employing one of the assay methods described herein. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally-occurring organic molecules, nucleic acid molecules, and components thereof.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the mapping of lin-8. [0100]
FIG. 2 is a schematic representation of the rescue of lin-8 by B0454.1. [0101]
FIG. 3 shows the sequence homology of LIN-8 (SEQ ID NO:1) with several other polypeptides in [0102] C. elegans (SEQ ID NOS:55-67).
FIG. 4 is a schematic representation of the mapping of lin-56. [0103]
FIG. 5 is a schematic representation of the rescue of lin-56 by ZK673.3. [0104]
FIG. 6 shows the LIN-56 polypeptide sequence (SEQ ID NO:3) as well as an alignment indicating the homology of an internal region of LIN-56 (SEQ ID NO:51) with several other [0105] C. elegans polypeptides (SEQ ID NOS:52-54).
FIG. 7 shows the sequence homology of the mbt repeats present in LIN-61 (SEQ ID NO:5) compared with mbt repeats from transcriptional repressors in other species (SEQ ID NOS:7-15). [0106]
FIG. 8 shows a sequence alignment between LIN-61 (SEQ ID NO:5) and predicted worm (SEQ ID NO:69) and human (SEQ ID NO:68) proteins of unknown function. [0107]
FIG. 9 shows that LIN-56 is localized to the nuclei of wild-type [0108] C. elegans embryos (Panels A-C), larvae (Panel D), and adults. The staining shown was obtained with affinity-purified and pre-adsorbed rabbit polyclonal antibody HM1923.
FIG. 10 shows that LIN-56 staining is absent in lin-56(n2728) embryos (Panels A-C), larvae (Panel D), and adults. The staining shown was obtained with affinity-purified and pre-adsorbed rabbit polyclonal antibody HM1923. [0109]
FIG. 11 shows that lin-61(RNAi) embryos display a failure in chromosome condensation during the first abortive mitotic division. [0110]
FIG. 12 shows that lin-61(RNAi) embryos display early embryonic lethality and a failure to complete cytokineses. [0111]
FIG. 13 shows that lin-61(RNAi) embryos arrest as multiply nucleated single cytoplasms.[0112]

DETAILED DESCRIPTION OF THE INVENTION

1. Introduction [0113]
We have cloned lin-8, lin-56, and lin-61 of the [0114] C. elegans synMuv gene family. lin-8 and lin-56 are class A genes, and lin-61 is a class B gene. These genes function in cell proliferation and are members of a tumor suppressor pathway, which is related to a tumor suppressor pathway of clinical importance in humans. Accordingly, the genes described herein, mutations in these genes, as well as the previously known synMuv genes, may be used to identify new tumor suppressors in other species, such as mammals, and may be used to identify therapeutic compounds. The encoded polypeptides, and nematodes carrying the newly cloned genes or mutations in these genes may similarly be employed.
lin-8 encodes a novel polypeptide. The invention provides the polypeptide sequence (SEQ ID NO:1), the nucleic acid sequence (SEQ ID NO:2), and lin-8 mutants, for example, lin-8(n2738), lin-8(n2731), lin-8(n3606), lin-8(n3595), lin-8(n2739), lin-8(n3586), lin-8(n3588), lin-8(n-111), lin-8(n2741), lin-8(n3585), lin-8(n3646), lin -8(n2376), lin-8(n2378), lin-8(n2403), lin-8(n2724), lin-8(n3607), lin-8(n3591), lin-8(n3609) and lin-8(n3581). [0115]
We also cloned lin-56 and describe the LIN-56 polypeptide sequence (SEQ ID NO:3) and the lin-56 nucleic acid sequence (SEQ ID NO:4) in the present invention, as well as lin-56 mutants, such as lin-56(n3355) and lin-56(n2728). [0116]
In addition, we cloned lin-61 (SEQ ID NO:6). The polypeptide encoded by this gene (SEQ ID NO:5) has homology with [0117] C. elegans and human polypeptides of unknown function, and to Drosophila polycomb group members and their related human proteins. Furthermore, we identified lin-61 mutants, for example, lin-61(n3446), lin-61(n3447), lin-61(n3624), and lin-61(n3635).
The present invention also provides mammalian homologs of the novel lin-8, lin-56, or lin-61 genes, which may be obtained using routine methods known to those skilled in the art. Such homologs may function in activating, enhancing, or otherwise intensifying the effects of tumor suppressors or oncogenes in mammals. [0118]
Genetic enhancer or suppressor screens may be performed to identify new genes that may function in initiating, enhancing, or otherwise interfacing with this tumor suppressor pathway. In addition, the identification of the lin-8, lin-56, or lin-61 genes, in combination with what is known about proliferative disease pathways in mammals, allows one skilled in the art to readily devise drug screens involving these genes to search for compounds that affect cell proliferation. Specifically, compounds that block the Muv phenotype of animals with a mutation in lin-8, lin-56, or lin-61 mutant animals are potential anti-tumor agents. The Muv phenotype may be present, for example, in a [0119] C. elegans with either a mutation in lin-8 or lin-56 in combination with a reduction of function mutation in a synMuv B gene, or a mutation in lin-61 in combination with a reduction of function mutation in a synMuv A gene. Compounds that stimulate cell division in animals with a single, silent lin-8, lin-56, or lin-61 mutation are likely to be agonists of cell proliferation and may act in a manner analogous to growth factors.
By providing insight regarding the function of the lin-8, lin-56, or lin-61 members of the synMuv genes in tumor suppression, and by identifying mutants in these genes, we have provided, in concert with generally known molecular biology and nematode genetic methods, the necessary elements of such methods and the compounds required for the practice of such methods. [0120]
2. Cloning of lin-8 [0121]
The lin-8(n-111) allele was isolated in an EMS screen for cell-lineage mutants (Horvitz and Sulston, Genetics 96:435-454, 1980). The lin-8 gene was mapped to the eight-map-unit interval between sup-9 and lin-31 on chromosome II (Ferguson and Horvitz, Genetics 123:109-121, 1989). As there were no other cloned genes in this interval that could be used for finer mapping, we used deficiencies to more precisely locate lin-8 on the physical map. The left endpoints of three deletions, ccDf11, ccDf1, and ccDf2, that remove lin-31 but not sup-9 had previously been roughly located and we further mapped the left endpoint of ccDf1 using PCR techniques. Analysis of the phenotypes of the Dfheterozygotes revealed that lin-8 is only deleted by ccDf11, thus placing lin-8 between sup-9 and M151 (FIG. 1). [0122]

Further mapping against the polymorphism pPK5363 placed lin-8 between sup-9 and C17F4. We identified a pool of two cosmids in this region that rescued the lin-8(lf) synMuv phenotype in germline transformation experiments. We further determined that this rescue effect was attributable to the single cosmid C03E12. Germline transformation experiments with subclones of C03E12 indicated that lin-8 is encoded by the predicted gene B0454.1 (FIG. 2). RNAi of this open reading frame produces a class A synMuv phenotype. Furthermore, nineteen alleles of lin-8 have been sequenced and all alleles contain mutations within B0454.1 (Table 1).

TABLE 1


Mutations Identified in lin-8 Alleles

lin-8 allele	WT sequence	Mutant Sequence	Amino Acid Change

n2738	TGG	TAG	W79amber
n2731	CAA	TAA	Q113ochre
n3606	TGG	TGA	W147opal
n3595	TGG	TAG	W163amber
n3609	CAG	TAG	Q279amber
n2739	AGA	TGA	R304opal
n3586, n3588	CAA	TAA	Q340ochre
n111	CTG	CCG	L20P
n2741	GTG	ATG	V68M
n3585	CGC	CAC	R127H
n3646	CGC	CAC	R146H
n2376	GAG	AAG	E148K
n2378	CGC	TGC	R154C
n2403, n2724, n3607	GAG	AAG	E164K
n3591	GAG	AAG	E347K
n3581	GTG	-TG	frameshift after aa192

The LIN-8 polypeptide is 386 amino acids in length, is novel, and appears to be highly charged. However, the LIN-8 polypeptide shares sequence homology with several other [0124] C. elegans polypeptides (FIG. 3).
3. Cloning of lin-56 [0125]
Identified as part of a screen to isolate new synMuv class A genes, lin-56 was previously mapped to the three-map-unit interval between dpy-10 and unc-53, close to unc-4, on chromosome II. We used the numerous deficiencies available in this region to further delineate the physical position of lin-56. The phenotypes of the Dfheterozygotes suggested that lin-56 is positioned between the cloned markers stP98 and bli-1 (FIG. 4). We identified a pool of five cosmids in this region that rescued the lin-56(lf) synMuv phenotype in germline transformation experiments using a lin-56(n2728);lin-15B(n744) strain. This rescue effect was attributed to the single cosmid ZK673 (FIG. 5). [0126]
Further germline transformation experiments with ZK673 subclones limited the lin-56 candidates to two predicted genes, ZK673.3 and ZK673.4. Using a combination of Southern blotting and PCR techniques, we determined that lin-56(n2728) contains an 11.2 kb deletion that completely eliminates these two genes and also removes the 3′ end of a third gene, ZK673.2. RNAi of ZK673.3, but not ZK673.4, produced a synMuv phenotype in a lin-15B(n744) background. [0127]
We have also identified a second lin-56 allele, designated lin-56(n3355), isolated in a lin-56(n2728) non-complementation screen. Sequencing of this gene revealed that lin-56(n3355) contains a stop codon in ZK673.3, confirming our identification of this open reading frame as lin-56. [0128]
The LIN-56 polypeptide is 322 amino acids in length (SEQ ID NO:3). This polypeptide appears to be novel and highly charged. LIN-56 does have an internal sequence (SEQ ID NO:51) that shares weak similarities with sequences in ZK673.4, LIN-15A, and a predicted polypeptide T25B9.8 (SEQ ID NOS:52-54)(Fig. 6). This region (SEQ ID NO:51) contains a C3H motif, which consists of a series of three cysteines followed by a histidine (C-x(2)-C-x(16)-A-x(7)-V-x(9)-A-x(11)-C-x(2)-H), where “x” can be any amino acid and where the number in parentheses indicates the number of “x” amino acids present at that position of the motif. The C3H motif suggests the presence of a metal binding domain, for example, a zinc-finger domain. However, the spacing of these residues is unlike that seen in any of the traditional motifs of this type. [0129]
In addition, antibodies to LIN-56 have been generated, using the full length LIN-56 polypeptide to form a fusion protein with glutathione S-transferase. These antibodies were purified against a fusion between full length LIN-56 and maltose binding protein (MBP). The antibodies recognize the LIN-56 polypeptide in nematode extracts, as assessed by Western blot analysis. [0130]
4. Cloning of lin-61 [0131]
We cloned the class B synthetic multivulva gene lin-61 by means of mapping, transformation rescue with cosmid R06C7, and the direct sequencing of multiple mutant alleles (see below). The cDNA sequence was determined based on the intron/exon structure of the genomic DNA. The predicted cDNA contains an SL1 splice leader sequence. Based upon a combination of sequence from the 5′ and 3′ ends of the cDNAs, and the genomic sequence of predicted ORF R06C7.7, we found lin-61 to encode a protein highly similar to predicted human and [0132] C. elegans proteins of undetermined function (FIG. 8).
The predicted LIN-61 protein contains motifs associated with transcriptional repressor proteins in species ranging from Drosophila to human, indicating that LIN-61 may play a similar role in transcriptional repression. The predicted LIN-61 protein contains four mbt repeats. These motifs are present in members of the [0133] Drosophila melanogaster polycomb family of transcriptional repressor proteins, including the proteins lethal(3) malignant brain tumor (1(3)mbt), and sex combs on the midleg (SCM). These motifs are also present in homologs of polycomb family members in other species, including human (FIG. 7). Analysis of mutant SCM alleles in Drosophila suggests that these mbt repeats are essential for SCM function (Bowermann et al., Genetics 150:675-686, 1998).
In addition, we found lin-61 to be 41.1% identical at the nucleotide level to Drosophila 1(3)mbt, and to be 40.8% identical at the nucleotide level to the human 1(3)mbt homolog. Homology at the protein level, however, is much greater within the mbt repeats. [0134]
We have isolated five alleles of lin-61 in addition to the mutation sy223 that originally defined the gene. A total of five alleles (including sy223) have been sequenced in our lab. sy223 is a single base pair alteration (G to A at position 2228 of SEQ ID NO:76) altering the splice acceptor site prior to the final exon of lin-61. lin-61(n3446) is single base pair alteration (C to T at position 1234 of SEQ ID NO:6) causing an in frame ochre stop codon in place of Glutamine 413 of SEQ ID NO:5 and lin-61 (n3447) is a single base pair alteration (G to A at position 1061 of SEQ ID NO:6) causing an Serine to Asparagine missense mutation at [0135] amino acid 355 of SEQ ID NO:5. Furthermore, we identified lin-61(n3624) to be a single base pair alteration (C to T at position 394 of SEQ ID NO:6) that causes a Proline to Serine missense mutation at amino acid 133 of SEQ ID NO:5 and lin-61(n3635) to encompass a single base pair alteration (G to A at position 1137 of SEQ ID NO:76) in the splice acceptor site prior to the putative fourth exon of the protein.
Each of these alleles causes a synthetic multivulva phenotype in combination with a class A synMuv gene. This is the only phenotype we have discovered to be associated with these mutations, and the allele lin-61(n3446) has wild-type vulval morphology when not in combination with a class A synMuv gene. [0136]
None of the lin-61 alleles described above represent a clear molecular null. We have therefore sought to determine the phenotype associated with complete loss of lin-61 function by means of RNA-mediated gene interference (RNAi). When wild-type animals or animals containing a mutation in a class A synMuv gene are injected with dsRNA synthesized from cDNA corresponding to a portion of lin-61, they produce progeny which suffer from completely penetrant embryonic lethality. This phenotype includes an inability to complete cytokinesis beginning at the single cell stage (FIG. 12A). In these embryos, the cytokinetic furrow forms and, at first, ingresses essentially normally (FIG. 12B-D), but subsequently regresses (FIG. 12E-G), resulting in a polyploid single cytoplasm (FIG. 12H). DNA replication continues to occur in the absence of completed cytokinesis, and the embryos display a terminal phenotype in which high levels of DNA (as visualized by DAPI staining (FIG. 13B)) and multiple mitotic spindles (as visualized by anti-tubulin antibody staining (FIG. 13C)) are present within what is often a single cytoplasm (FIG. 13A). Moreover, embryos from lin-61(RNAi) mothers (FIG. 11A) display a failure to condense mitotic chromosomes (as visualized by DAPI staining (FIG. [0137] 11B)). In these embryos, the mitotic spindle was visualized with an anti-tubulin antibody (FIG. 11C).
We further examined the developmental functions of lin-61 by injecting lin-61 dsRNA into RNAi defective rde-1 hermaphrodites and then mating these animals with N2 males (a technique described by Herman, Development 128:581-590, 2001). Cross progeny were then observed in an effort to gain an understanding of the phenotype produced after the reduction of zygotic lin-61 activity, while maintaining at least some maternal lin-61 function. Unlike lin-61(RNAi), this approach yields animals that reach adulthood but display a host of developmental abnormalities including uncoordinated movement, abnormal development of the male tail, and possible abnormalities in the structure of the vulva. [0138]
Based on these results we conclude that lin-61 functions in a variety of embryonic and post-embryonic developmental processes in addition to its role in vulval development and cell proliferation. [0139]
5. Characterization of Interactions among the lin-8, lin-56, lin-61 and Other synMuv Gene Products [0140]
Standard yeast two-hybrid techniques are used to characterize the physical interactions between the lin-8, lin-56, lin-61 and other synMuv gene products, for example, as described in U.S. Ser. No. 09/220,091, incorporated herein by reference. These two-hybrid systems can also be used to detect therapeutic compounds, which disrupt the synMuv protein-protein interactions, including interactions between lin-8, lin-56, and tin-61 gene products and other synMuv gene products. For example, in a genome-wide yeast two-hybrid screen, using LIN-35 as a bait, we showed LIN-8 and LIN-35 to interact. [0141]
Interactions among the lin-8, lin-56, lin-61 and other synMuv gene products can also be examined using other methods known to one skilled in the art. For example such interactions can be determined by performing GST pull-down experiments using the appropriate fusion proteins, as described in U.S. Ser. No. 09/220,091. Alternatively, interactions may be further investigated through the use of triple mutants using the appropriate genes, as also described in U.S. Ser. No. 09/220,091. [0142]
6. Non-Vulval Phenotypes in lin-8 and lin-61 Mutants [0143]
lin-8 and lin-6 are also involved in a non-vulval phenotype. During the course of a [0144] C. elegans screen using a cell-type specific reporter, which results in the expression of green fluorescent protein, we discovered that a class of mutants exists in which this reporter is ectopically expressed in pharyngeal tissue, especially in the posterior pharynx. We refer to this phenotype to as the “green pharynx phenotype.” Further studies have revealed that loss-of-function mutations in any of several genes (lin-8, lin-13, lin-61) belonging to both the class A and class B synMuv pathways can cause the green pharynx phenotype. In the case of lin-8, all but one allele (n2376, E148K) tested exhibit the green pharynx phenotype, providing a single, distinct loss-of-function phenotype for this gene.
In addition, some of the synMuv B genes are homologues of NuRD complex components and loss-of-function mutations in other components of the NuRD chromatin remodeling complex (e.g., lin-40/egr-1 and chd-3) can also cause the green pharynx phenotype. Furthermore, the green pharynx phenotype has been observed with two distinct cell fate-specific reporters (pkd-2::gfp and lin-11::gfp). [0145]
The green pharynx phenotype is observed irrespective of whether the reporter transgene is integrated into the genome or whether it is present extra-chromosomally, indicating that chromosomal integration is not required. Additionally, when using an integrated reporter, the phenotype does not appear to be highly dependent on the site of chromosomal integration and the phenotype is observed with both low and high copy number transgenes. [0146]
Our data indicate that the synMuv A and B genes may act in similar processes, and may work together in contexts other than vulval development and provide additional evidence that lin-8, lin-13 and lin-61 act in transcriptional repression. [0147]
7. Cloning lin-8, lin-56 and lin-61 Vertebrate Genes [0148]
The invention described herein provides the identity of class A synMuv genes lin-8 and lin-56, and the class B synMuv gene lin-61. In view of what is know in the field regarding the synMuv family and its relationship to related genes in the Rb tumor suppressor pathway, we conclude that these newly cloned genes are also involved in pathways that modulate tumor suppression. It is likely that [0149] C. elegans lin-8, lin-56 and lin-61 genes have ortholog counterparts in vertebrates, for example mammalian genes. One skilled in the art will recognize that these orthologs can be identified using standard techniques in molecular biology, such as screening of cDNA or genomic libraries, degenerate PCR, and the like, described in, for example, Ausubel et al. (supra). Orthologs can also be identified using computer-based search programs such as BLAST and dbEST to isolate expressed sequence tags (ESTs) with regions of similarity or identity to a lin-8, lin-56 or lin-61 gene.
Vertebrate counterparts of [0150] C. elegans lin-8, lin-56 and lin-61 are candidate tumor suppressor genes. Thus, one can screen for mutations in the human homologs of these genes in patients diagnosed with cancer or in immortalized cell lines. Similarly, the polypeptides encoded by these genes are candidate targets for anti-cancer drugs. A drug which increases synMuv polypeptide activity, for example, LIN-8, LIN-56, or LIN-61 biological activity, may decrease proliferation of tumor cells. In addition, polypeptides which interact with other synMuv polypeptides or which regulate synMuv gene expression are also candidate tumor suppressors; these polypeptides can be isolated using standard techniques, as described herein or, for example, in Ausubel et al. (supra).
8. LIN-8, LIN-56, or LIN-61 Polypeptide Expression [0151]
A lin-8, lin-56, or lin-61 nucleic acid sequence may be expressed in a prokaryotic or eukaryotic cell. In addition, it may be desirable to express the nucleic acid sequence under the control of an inducible promoter for the purposes of polypeptide production. [0152]
In general, LIN-8, LIN-56, or LIN-61 polypeptides may be produced by transformation of a suitable host cell with all or part of a LIN-8, LIN-56, or LIN-61-encoding cDNA fragment (e.g., the cDNAs described above) in a suitable expression vehicle. [0153]
Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. The LIN-8, LIN-56, or LIN-61 polypeptide may be produced in a prokaryotic host (e.g., [0154] E. coli ) or in a eukaryotic host (e.g., nematodes, Saccharomyces cerevisiae, insect cells, e.g., Sf-21 cells, or mammalian cells, e.g., COS 1, NIH 3T3, or HeLa cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al. (supra)). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
One preferred expression system is the baculovirus system (using, for example, the vector pBacPAK9) available from Clontech (Palo Alto, Calif.). If desired, this system may be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985). [0155]
Alternatively, a LIN-8, LIN-56, or LIN-61 polypeptide is produced by a stably-transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra). In one example, cDNA encoding a LIN-8, LIN-56, or LIN-61 polypeptide is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid, and, therefore, the LIN-8, LIN-56, or LIN-61 polypeptide-encoding gene, into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (as described in Ausubel et al. (supra)). This dominant selection can be accomplished in most cell types and recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. In addition, methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra); such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al. (supra)). Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR[0156] ⁽⁻⁾cells, ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification.
Once the recombinant LIN-8, LIN-56, or LIN-61 protein is expressed, it is isolated, e.g., using affinity chromatography. In one example, an anti-LIN-8, LIN-56, or LIN-61 protein antibody (e.g., produced as described herein) may be immobilized on a column and used to isolate the LIN-8, LIN-56, or LIN-61 protein. Lysis and fractionation of LIN-8, LIN-56, or LIN-61 protein-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al. (supra)). [0157]
Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high pressure liquid chromatography (see, e.g., Fisher, [0158] Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
Polypeptides of the invention, particularly short LIN-8, LIN-56, or LIN-61 protein fragments, can also be produced by chemical synthesis (e.g., by the methods described in [0159] Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
These general techniques of polypeptide expression and purification can also be used to produce and isolate useful LIN-8, LIN-56, or LIN-61 fragments or analogs (described herein). [0160]
9. Anti-LIN-8, LIN-56 or LIN-61 Antibodies [0161]
In general, to generate a LIN-8, LIN-56, or LIN-61-specific antibody, a lin-8, lin-56, or lin-61 coding sequence may be expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31-40, 1988). The fusion protein can be purified on glutathione-sepharose beads, eluted with glutathione cleaved with thrombin (at the engineered cleavage site), and purified to the degree necessary for immunization of rabbits. Primary immunizations can be carried out with Freund's complete adjuvant and subsequent immunizations with Freund's incomplete adjuvant. Antibody titres are monitored by Western blot and immunoprecipitation analyses using the thrombin-cleaved LIN-8, LIN-56, or LIN-61 polypeptide fragment of the GST-LIN-8, -LIN-56, or -LIN-61 fusion protein. Immune sera are affinity purified using, for example, CNBr-Sepharose-coupled LIN-8, LIN-56, or LIN-61 protein. Antiserum specificity is determined using a panel of unrelated GST proteins (including GSTp53, Rb, HPV-16 E6, and E6-AP) and GST-trypsin (which was generated by PCR using known sequences). [0162]
As an alternate or adjunct immunogen to GST fusion proteins, peptides corresponding to relatively unique regions of LIN-8, LIN-56, or LIN-61 may be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine. Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity tested in ELISA and Western blots assays using peptide conjugates, and by Western blot and immunoprecipitation techniques using LIN-8, LIN-56, or LIN-61 expressed as a GST fusion protein. [0163]
Alternatively, monoclonal antibodies may be prepared using the LIN-8, LIN-56, or LIN-61 proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In [0164] Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; Ausubel et al., (supra)). Once produced, monoclonal antibodies are also tested for specific recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al. (supra). Antibodies which specifically recognize LIN-8, LIN-56, or LIN-61 are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of LIN-8, LIN-56, or LIN-61 produced by an animal (for example, to determine the amount or subcellular location of LIN-8, LIN-56, or LIN-61).
Preferably antibodies of the invention are produced using fragments of the LIN-8, LIN-56, or LIN-61 polypeptide that are positioned outside highly conserved regions and appear likely to be antigenic, by criteria such as those provided by the Peptidestructure program of the Genetics Computer Group Sequence Analysis Package (Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988). In one specific example, such fragments are generated by standard PCR techniques and cloned into the pGEX expression vector (Ausubel et al. (supra). Fusion proteins are expressed in [0165] E. coli and purified using a glutathione agarose affinity matrix, as described in Ausubel et al. (supra). To attempt to minimize the potential problems of low affinity or specificity of antisera, two or three such fusions are generated for each polypeptide, and each fusion is injected into at least two rabbits. Antisera are raised by injections in a series, preferably including at least three booster injections.
To demonstrate the utility of this approach, we generated polyclonal antibodies against a fusion of full-length LIN-8 with maltose binding protein (MBP) (see Example 1 for the detailed protocol used). In order to increase the sensitivity of the antibodies, they were affinity-purified against a GST::LIN-8 fusion, and pre-adsorbed with extract from lin-8(n2731) worms. [0166]
In addition, the antibodies that we generated against LIN-56 (see Example 2) were also affinity purified and pre-adsorbed with extract from lin-56(n2728) worms. We used one of these anti-LIN-56 antibodies (HM1923) for Western analysis and for wholemount staining. This antibody recognizes a doublet in wild-type but not lin-56(n2728) worm extracts on Western analysis and the proteins in this doublet fractionate specifically with nuclear material. Furthermore, wholemount staining with this antibody reveals that lin-56 is expressed in the nuclei of most if not all cells throughout development and adulthood (FIG. 9 A-D). We also stained lin-56(n2728) embryos, larvae, and adults to show that the HM1923 antibody is specific for LIN-56. As is seen in FIG. 10, LIN-56 staining is absent in LIN-56 mutant worms, indicating that the antibody is specific for LIN-56. [0167]
In addition, LIN-56 appears to be absent from nuclei during part of the cell cycle, probably as a result of nuclear membrane breakdown. Furthermore, LIN-56 expression and localization appear wild-type in the synMuv A lin-8 and lin-38 mutants, but the nuclear expression of LIN-56 appears severely reduced or even absent in lin-15A(n767) and lin-15AB(n309) mutants. By Western analysis, LIN-56 protein levels do in fact appear reduced in lin-15A(n767) worm extracts, and the ratio of the bands in the detected doublet may even be altered. [0168]
10. Identification of Molecules that Modulate LIN-8, LIN-56, or LIN-61 Polypeptide Expression [0169]
Isolation of lin-8, lin-56, or lin-61 cDNAs also facilitates the identification of molecules which increase or decrease LIN-8, LIN-56, or LIN-61 expression. According to one approach, candidate molecules are added at varying concentrations to the culture medium of cells or nematodes expressing LIN-8, LIN-56, or LIN-61. lin-8, lin-56, or lin-61 expression is then measured, for example, by standard Northern blot analysis (Ausubel et al. (supra)) using a lin-8, lin-56, or lin-61 cDNA (or cDNA fragment) as a hybridization probe (see also Table III). The level of lin-8, lin-56, or lin-61 expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule. When nematodes are being used, the phenotypes associated with the synMuv pathway may be utilized as the primary screen for alteration in polypeptide expression. [0170]
If desired, the effect of candidate modulators on expression may, in the alternative, be measured at the level of LIN-8, LIN-56, or LIN-61 polypeptide production using the same general approach and standard immunological detection techniques, such as Western blotting or immunoprecipitation with a LIN-8, LIN-56, or LIN-61-specific antibody (for example, the LIN-8, LIN-56, or LIN-61 antibody described herein). [0171]
Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al. (supra)). In a mixed compound assay, LIN-8, LIN-56, or LIN-61 expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to modulate LIN-8, LIN-56, or LIN-61 expression. [0172]
Alternatively, or in addition, candidate compounds may be screened for those, which modulate LIN-8, LIN-56, or LIN-61 cell proliferation. In this approach, the degree of cell proliferation, or the LIN-8, LIN-56, or LIN-61 phenotype in the presence of a candidate compound is compared to the degree of cell proliferation in its absence, under equivalent conditions. Again, such a screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion. Cell proliferation may be measured by any standard assay. [0173]
Candidate LIN-8, LIN-56, or LIN-61 modulators include peptide as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured). [0174]
Modulators found to be effective at the level of LIN-8, LIN-56, or LIN-61 expression or biological activity may be confirmed as useful in animal models and, if successful, may be used as anti-cancer therapeutics to increase or decrease cell proliferation. [0175]
11. lin-8, lin-56, or lin-61 Therapy [0176]
Because expression levels of lin-8, lin-56, or lin-61 genes correlate with the levels of cell proliferation, such genes also find use in gene therapy to modulate cell proliferation. [0177]
Retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or other viral vectors with the appropriate tropism for cells likely to be involved in the cell proliferation disease may be used as a gene transfer delivery system for a therapeutic lin-8, lin-56, or lin-61 gene construct. Numerous vectors useful for this purpose are generally known in the art (Miller, Human Gene Therapy 5-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and Anderson, Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; and Miller and Rosman, BioTechniques 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). [0178]
Non-viral approaches may also be employed for the introduction of therapeutic DNA into cells otherwise predicted to undergo insufficient or excess cell proliferation. For example, lin-8, lin-56, or lin-61 may be introduced into a cell by the techniques of lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Lett. 117:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger and Papahadjopoulos, Meth. Enz. 101:512, 1983); asialorosonucoid-polylysine conjugation (Wu and Wu, J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or, less preferably, microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). [0179]
For any of the above approaches, the therapeutic lin-8, lin-56, or lin-61 DNA construct, or an antisense nucleic acid, is preferably applied to the site of the predicted cell proliferation event (for example, by injection), but may also be applied to tissue in the vicinity of the predicted event or even to a blood vessel supplying the cells predicted to undergo insufficient or excess cell proliferation. [0180]
In the gene therapy constructs, lin-8, lin-56, or lin-61 cDNA expression is directed from any suitable promoter (e.g., the human cytomegalovirus, [0181] simian virus 40, or metallothionein promoters), and its production is regulated by any desired regulatory element. For example, if desired, enhancers known to direct preferential gene expression in a particular cell may be used to direct lin-8, lin-56, or lin-61 expression. Such enhancers include, without limitation, those enhancers which are characterized as tissue or cell specific in their expression.
Alternatively, if a lin-8, lin-56, or lin-61 genomic clone is utilized as a therapeutic construct (for example, following its isolation by hybridization with the lin-8, lin-56, or lin-61 cDNA described above), lin-8, lin-56, or lin-61 expression is regulated by its cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, e.g., any of the promoters or regulatory elements described above. [0182]
Less preferably, lin-8, lin-56, or lin-61 gene therapy is accomplished by direct administration of the lin-8, lin-56, or lin-61 mRNA to a cell predicted to undergo excess or insufficient cell proliferation. This mRNA may be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using a lin-8, lin-56, or lin-61 cDNA under the control of a high efficiency promoter (e.g., the T7 promoter). Administration of lin-8, lin-56, or lin-61 mRNA to malignant cells is carried out by any of the methods for direct nucleic acid administration described above. [0183]
Ideally, the production of a LIN-8, LIN-56, or LIN-61 polypeptide by any gene therapy approach described above results in a cellular level of LIN-8, LIN-56, or LIN-61 that is at least equivalent to the normal, cellular level of LIN-8, LIN-56, or LIN-61 in an unaffected individual. Treatment by any lin-8, lin-56, or lin-61 -mediated gene therapy approach may be combined with more traditional therapies. [0184]
Another therapeutic approach included within the invention involves direct administration of recombinant LIN-8, LIN-56, or LIN-61 protein, either to the site of a predicted or desirable cell proliferation event (for example, by injection) or systemically by any conventional recombinant protein administration technique. The actual dosage of LIN-8, LIN-56, or LIN-61 administered depends on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 100 mg, inclusive, are administered per day to an adult in any pharmaceutically-acceptable formulation. [0185]
The nucleic acids of the present invention may also be utilized in plant cells. Such sequences may be expressed in plant cells, and used, for example, to promote plant survival or growth (e.g., by providing disease resistance). [0186]
12. Administration of LIN-8, LIN-56, or LIN-61 Polypeptides, lin-8, lin-56, or lin-61 Nucleic Acid Sequences, or Modulators of LIN-8, LIN-56, or LIN-61 Synthesis or Function [0187]
A LIN-8, LIN-56, or LIN-61 polypeptide, nucleic acid sequence, or modulator may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer LIN-8, LIN-56, or LIN-61 to patients suffering from, or presymptomatic for, a LIN-8, LIN-56, or LIN-61, or synMuv-associated cancer. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. [0188]
Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences ((18[0189] ^thedition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for LIN-8, LIN-56, or LIN-61 polypeptides, nucleic acid sequences or modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
If desired, treatment with a LIN-8, LIN-56, or LIN-61 polypeptide, nucleic acid sequence, or modulatory compound may be combined with more traditional therapies for the disease such as surgery, radiation, or chemotherapy for cancers. [0190]
13. Detection of A Condition Involving Altered Cell Proliferation or an Increased Likelihood of Developing a Cell Proliferation Disease [0191]
LIN-8, LIN-56, or LIN-61 polypeptides and nucleic acid sequences find diagnostic use in the detection or monitoring of conditions involving aberrant levels of cell proliferation. A decrease or increase in the level of LIN-8, LIN-56, or LIN-61 production may provide an indication of a deleterious condition. Levels of LIN-8, -56, or LIN-61 expression may be assayed by any standard technique. For example, its expression in a biological sample (e.g., a biopsy) may be monitored by standard Northern blot analysis, using, for example, probes designed from lin-8, lin-56, or lin-61 nucleic acid sequences, or from nucleic acid sequences that hybridize to a lin-8, lin-56, or lin-61 nucleic acid sequence. Measurement of such expression may be aided by PCR (see, e.g., Ausubel et al. (supra); [0192] PCR Technology: Principles and Applications for DNA Amplification, ed., H. A. Ehrlich, Stockton Press, NY; and Yap and McGee, Nucl. Acids Res. 19:4294, 1991).
Alternatively, a patient sample may be analyzed for one or more mutations in the lin-8, lin-56, or lin-61 sequences using a mismatch detection approach. Generally, these techniques involve PCR amplification of nucleic acid from the patient sample, followed by identification of the mutation (i.e., mismatch) by either altered hybridization, aberrant electrophoretic gel migration, binding or cleavage mediated by mismatch binding proteins, or direct nucleic acid sequencing. Any of these techniques may be used to facilitate mutant lin-8, lin-56, or lin-61 detection, and each is well known in the art (see, for example, Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989; and Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989). [0193]
In yet another approach, immunoassays are used to detect or monitor a LIN-8, LIN-56, or LIN-61 polypeptide in a biological sample. LIN-8, LIN-56, or LIN-61-specific polyclonal or monoclonal antibodies (produced as described above) may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure LIN-8, LIN-56, or LIN-61 polypeptide levels; again comparison is to wild-type LIN-8, LIN-56, or LIN-61 levels, and an increase or decrease in LIN-8, LIN-56, or LIN-61 production is indicative of a condition involving altered cell proliferation. Examples of immunoassays are described, e.g., in Ausubel et al. (supra). Immunohistochemical techniques may also be utilized for LIN-8, LIN-56, or LIN-61 detection. For example, a tissue sample may be obtained from a patient, and a section stained for the presence of LIN-8, LIN-56, or LIN-61 using an anti-LIN-8, LIN-56, or LIN-61 antibody and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens ([0194] Theory and Practice of Histological Techniques, Churchill Livingstone, 1982) and Ausubel et al. (supra).
In one preferred example, a combined diagnostic method may be employed that begins with an evaluation of LIN-8, LIN-56, or LIN-61 polypeptide production (for example, by immunological techniques or the protein truncation test (Hogerrorst et al., Nature Genetics 10:208-212, 1995) and also includes a nucleic acid-based detection technique designed to identify more subtle lin-8, lin-56, or lin-61 mutations (for example, point mutations). As described above, a number of mismatch detection assays are available to those skilled in the art, and any preferred technique may be used (see above). By this approach, mutations in lin-8, lin-56, or lin-61 may be detected that either result in loss of LIN-8, LIN-56, or LIN-61 expression or biological activity. [0195]
Mismatch detection assays also provide the opportunity to diagnose a lin-8, lin-56, or lin-61-mediated predisposition to diseases of cell proliferation. For example, a patient heterozygous for a lin-8, lin-56, or lin-61 mutation may show no clinical symptoms and yet possess a higher than normal probability of developing one or more types of diseases. Given this diagnosis, a patient may take precautions to minimize their exposure to adverse environmental factors (for example, UV exposure or chemical mutagens) and to carefully monitor their medical condition (for example, through frequent physical examinations). This type of lin-8, lin-56, or lin-61 diagnostic approach may also be used to detect lin-8, lin-56, or lin-61 mutations in prenatal screens. [0196]
The lin-8, lin-56, or lin-61 diagnostic assays described above may be carried out using any biological sample (for example, any biopsy sample or bodily fluid or tissue) in which lin-8, lin-56, or lin-61 is normally expressed. Identification of a mutant lin-8, lin-56, or lin-61 gene may also be assayed using these sources for test samples. Alternatively, a lin-8, lin-56, or lin-61 mutation, particularly as part of a diagnosis for predisposition to lin-8, lin-56, or lin-61-associated proliferative disease, may be tested using a DNA sample from any cell, for example, by mismatch detection techniques; preferably, the DNA sample is subjected to PCR amplification prior to analysis. [0197]
The following examples are meant to illustrate the invention and should not be construed as limiting. [0198]

EXAMPLES

Example 1

Generation of Rabbit and Guinea Pig Polyclonal Antibodies Against LIN-8

We made a fusion protein of full-length LIN-8 with MBP and had Covance (Richmond, Calif.) produce LIN-8 polyclonal antibodies using two rabbits and two guinea pigs. However, anyone skilled in the art may generate antibodies against LIN-8 using the following protocol. [0199]
New Zealand White Female Rabbits were bled prior to injection of the antigen, for later use as a control in establishing background reactivity of the serum. Following the prebleed, the rabbits were injected subcutaneously at multiple sites with 250 μg protein and 0.5mL Freund's Complete Adjuvant (FCA). Three weeks later, the rabbits received a subcutaneous, dorsal, boost injection of 125 μg protein with 1.0 mL Freund's Incomplete Adjuvant (FIA). The first test bleed was performed 11 days later, followed by a second subcutaneous, dorsal boost injection of 125 μg protein and 10 [0200] mL FIA 9 days after the test bleed. A second test bleed was performed 11 days after the second boost, followed by a third subcutaneous, dorsal boost of 125 μg protein and 1.0 mL FIA 10 days after the second test bleed. The first production bleed was performed 10 days after the third boost, followed by a fourth subcutaneous, dorsal boost of 125 μg and 1.0 mL FIA 10 days after the first production bleed. A second production bleed was performed 11 days after the fourth boost, followed by a fifth subcutaneous, dorsal boost of 125 μg and 1.0 mL FIA 10 days after the second production bleed. A third production bleed was performed 11 days after the fifth boost, followed by exsanguination after 6 days.
The protocol used to produce LIN-8 antibodies in guinea pigs closely follows the one outlined for rabbits above. Dunkin Hartley Guinea Pig were used and prebled prior to subcutaneous and intradermal injection of 200 μg protein and 1.0 mL FCA. Three weeks after the primary injection, the guinea pigs received a boost injection of 100 μg protein and 0.5 mL FIA, subcutaneously in the neck. After 11 days, the first test bleed was performed, followed 10 days later by a second boost of 100 μg protein and 0.5 mL FIA, subcutaneously in the neck. The second test bleed was performed after 11 days, followed 10 days later by a third boost of 100 μg protein and 0.5 mL FIA, subcutaneously in the neck. The first production bleed was performed 11 days after the third boost, followed 10 days later by a fourth subcutaneous, dorsal boost of 100 μg protein and 0.5 mL FIA. The second production bleed followed 11 days later and a fifth boost of 100 μg protein and 0.5 mL FIA was performed after 10 days. The third production bleed was performed 11 days later, followed by exsanguination after 6 days. [0201]
Generally, polyclonal antibodies are affinity purified to increase their specificity. In addition, the polyclonal antibody may be depleted of any components that do not specifically bind to the protein of interest by pre-adsorbing the antibody with an extract made from tissue that lacks the protein of interest. For example, an extract from lin-8(n2731) worms may be used to remove any antibodies that bind worm proteins besides LIN-8. [0202]

Example 2

Generation of Rabbit and Rat Polyclonal Antibodies Against LIN-56

We made a fusion protein of full-length LIN-56 with GST and had Covance (Richmond, Calif.) produce LIN-56 polyclonal antibodies using two rabbits and two rats. However, anyone skilled in the art may generate antibodies against LIN-56 using the following protocol. [0203]
New Zealand White Female Rabbits were bled prior to intradermal injection in the back with 250 μg protein and 0.5 mL FCA. Three weeks later, the rabbits received a subcutaneous nodal (groin and pit) area boost injection of 125 μg protein with 0.5 mL FIA. The first test bleed was performed 10 days later, followed by a second subcutaneous boost injection of 125 μg protein and 0.5 mL FIA in the neck, 11 days after the test bleed. A second test bleed was performed 10 days after the second boost, followed by a third subcutaneous, dorsal boost of 125 μg protein and 1.0 [0204] mL FIA 11 days after the second test bleed. The first production bleed was performed 10 days after the third boost, followed by a fourth subcutaneous nodal area (groin and pit) boost of 125 μg and 1.0 mL FIA 10 days after the first production bleed. A second production bleed was performed 11 days after the fourth boost, followed by a fifth subcutaneous, dorsal boost of 125 μg and 1.0 mL FIA 11 days after the second production bleed. A third production bleed was performed 10 days after the fifth boost, followed by exsanguination after 10 days.
The protocol used to produce LIN-56 antibodies in rats closely follows the one outlined for rabbits above. SD rats were used and prebled prior to subcutaneous injection of 200 μg protein and 0.4 mL FCA, in the neck. Three weeks after the primary injection, the rats received a boost injection of 100 μg protein and 0.4 mL FIA, subcutaneously in the neck. After 10 days, the first test bleed was performed, followed 11 days later by a second boost of 100 μg protein and 0.4 mL FIA, subcutaneously in the neck. The second test bleed was performed after 10 days, followed 11 days later by a third boost of 100 μg protein and 0.4 mL FIA, subcutaneously in the neck. The first production bleed was performed 10 days after the third boost, followed 11 days later by a fourth subcutaneous, dorsal boost of 100 μg protein and 0.4 mL FIA. The second production bleed followed 10 days later and a fifth boost of 100 μg protein and 0.4 mL FIA was performed after 11 days. The third production bleed was performed 10 days later, followed by exsanguination after 10 days. [0205]

Other Embodiments

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and which may be applied to the essential features hereinbefore set forth. [0206]
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. [0207]
1 78 1 386 PRT Caenorhabditis elegans 1 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 2 1276 DNA Caenorhabditis elegans 2 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 3 322 PRT Caenorhabditis elegans 3 Met Asp His His Ala Met Tyr Arg Thr Ala Glu Phe Asn Lys Thr Thr 1 5 10 15 Val Arg Leu Leu Ala Glu Phe Ile Glu Lys Thr Gly Gln Asn Ala Thr 20 25 30 Ile Val Asn Met Asp Ser Phe Leu Glu Phe Phe Ala Tyr Leu Asn Pro 35 40 45 Thr Ala Pro Ile Pro Thr Val Pro Glu Ile Glu Lys Gln Leu Leu Leu 50 55 60 Lys Ser Pro Ile Arg Cys Ile Val Cys Gly Met Glu Thr Glu Ser Asp 65 70 75 80 Ser Ala Val Thr Leu Ser Ile Asp Asn Ala Ser Ile Ile Leu Thr Ala 85 90 95 Thr Val Ile Gly Tyr Cys Arg Asp Pro Ser Asp Ala Val Asn Gln Ile 100 105 110 Arg Lys Glu Ser Leu Arg Ala Cys Thr Lys His Phe Asn Ser Ile Phe 115 120 125 His Val Ile Phe Glu Gly Leu Gln Ile Glu Asn Thr Tyr Cys Ala His 130 135 140 His Ala Lys Tyr Ser Leu Ala Asn Arg Trp Cys Lys Val Tyr Thr Met 145 150 155 160 Ile Arg Ser Ser Leu Gly Glu Gln Phe Thr Lys Phe Asp Val Arg Asn 165 170 175 Phe Lys Ser Ile Leu Gln Ser Phe Leu Asp Thr Phe Gly Glu Ile Asp 180 185 190 Asp Asp Lys Lys Asp Lys Glu Ser Ser His Phe Asp Glu Cys Phe Glu 195 200 205 Glu Met Asp Ser Glu Asn Val Glu Ile Lys Met Glu Ser Pro Gln Glu 210 215 220 Glu Ala Ala Glu Lys Ser Lys Phe Ser Glu Asn Leu Val Glu Val Lys 225 230 235 240 Leu Glu Pro Ile Glu Thr His Glu Leu Asp Lys Thr Ile Ser Asp Phe 245 250 255 Ser Ser Ser Asp Ile Ile Asp Ser Ser Gln Lys Leu Gln Gln Asn Gly 260 265 270 Phe Pro Glu Lys Val Glu Gln Met Asp Lys Tyr Ser Asn Lys Leu Lys 275 280 285 Asp Glu Ala Ser Asp Lys Lys Tyr Glu Lys Pro Gly Lys Lys Asp Tyr 290 295 300 Val Glu Glu Glu Gly Tyr Trp Ala Pro Ile Thr Asp Ser Glu Asp Asp 305 310 315 320 Glu Ala 4 1108 DNA Caenorhabditis elegans 4 gcaaaaaact agatattttg tggcattttt acaattaaaa aacctttaaa aaatggatca 60 ccatgctatg taccgaaccg ctgaattcaa caaaactact gtccgattat tggcggaatt 120 catcgaaaag actgggcaga atgcgacgat agtgaatatg gacagctttt tggagttctt 180 tgcgtatttg aatcccacgg ctccaattcc aacggttcca gaaattgaaa aacaattatt 240 gctaaaatca ccgattcgtt gcattgtgtg tggaatggaa actgaatcag attccgcagt 300 gacattaagc atcgataatg cttcaattat tctcacagcg acagtaattg gttactgtag 360 agatccaagt gatgcagtta atcaaattcg aaaggagagt cttcgagcat gcacgaaaca 420 tttcaacagt attttccatg tcatcttcga aggactgcaa atcgagaaca cctactgtgc 480 tcatcatgca aaatacagtc ttgccaatcg ttggtgcaaa gtctacacga tgattcgatc 540 ttccctgggc gagcagttca caaagttcga tgtgcgcaat tttaaatcaa tattgcaatc 600 atttttggat acttttggtg aaattgatga cgacaaaaag gataaagaat cttctcattt 660 tgatgaatgt tttgaagaaa tggattcaga aaacgtagaa attaaaatgg agagcccaca 720 agaagaagct gcagagaaat cgaagttttc tgaaaaccta gtggaggtaa aactggaacc 780 aattgaaact catgaacttg acaaaactat atccgacttt tcttcaagtg atataattga 840 ttcgtcccaa aaactgcagc aaaatggttt tcctgaaaaa gtggagcaaa tggacaaata 900 tagcaacaaa ttgaaagatg aagcttcaga caaaaagtat gaaaagccag gaaaaaagga 960 ctacgttgaa gaagagggat actgggcgcc gatcaccgac agcgaggatg atgaagcctg 1020 aatttattta atcaaacgtt ttggaaattt tttttgtttt tgtcaataaa accatataac 1080 aataaaaaaa aaaaaaaaaa aactcgag 1108 5 498 PRT Caenorhabditis elegans 5 Met Ser Glu Phe Leu Lys Ile Val Arg Ala Asn Lys Lys Ser Asp Arg 1 5 10 15 Lys Leu Asp Lys Thr Tyr Leu Trp Glu Ser Tyr Leu His Gln Phe Glu 20 25 30 Lys Gly Lys Thr Ser Phe Ile Pro Val Glu Ala Phe Asn Arg Asn Leu 35 40 45 Thr Val Asn Phe Asn Glu Cys Val Lys Glu Gly Val Ile Phe Glu Thr 50 55 60 Val Val His Asp Tyr Asp Lys Asn Cys Asp Ser Ile Gln Val Arg Trp 65 70 75 80 Phe Ala Arg Ile Glu Lys Val Cys Gly Tyr Arg Val Leu Ala Gln Phe 85 90 95 Ile Gly Ala Asp Thr Lys Phe Trp Leu Asn Ile Leu Ser Asp Asp Met 100 105 110 Phe Gly Leu Ala Asn Ala Ala Met Ser Asp Pro Asn Met Asp Lys Ile 115 120 125 Val Tyr Ala Pro Pro Leu Ala Ile Asn Glu Glu Tyr Gln Asn Asp Met 130 135 140 Val Asn Tyr Val Asn Asn Cys Ile Asp Gly Glu Ile Val Gly Gln Thr 145 150 155 160 Ser Leu Ser Pro Lys Phe Asp Glu Gly Lys Ala Leu Leu Ser Lys His 165 170 175 Arg Phe Lys Val Gly Gln Arg Leu Glu Leu Leu Asn Tyr Ser Asn Ser 180 185 190 Thr Glu Ile Arg Val Ala Arg Ile Gln Glu Ile Cys Gly Arg Arg Met 195 200 205 Asn Val Ser Ile Thr Lys Lys Asp Phe Pro Glu Ser Leu Pro Asp Ala 210 215 220 Asp Asp Asp Arg Gln Val Phe Ser Ser Gly Ser Gln Tyr Trp Ile Asp 225 230 235 240 Glu Gly Ser Phe Phe Ile Phe Pro Val Gly Phe Ala Ala Val Asn Gly 245 250 255 Tyr Gln Leu Asn Ala Lys Lys Glu Tyr Ile Glu His Thr Asn Lys Ile 260 265 270 Ala Gln Ala Ile Lys Asn Gly Glu Asn Pro Arg Tyr Asp Ser Asp Asp 275 280 285 Val Thr Phe Asp Gln Leu Ala Lys Asp Pro Ile Asp Pro Met Ile Trp 290 295 300 Arg Lys Val Lys Val Gly Gln Lys Phe Glu Leu Ile Asp Pro Leu Ala 305 310 315 320 Gln Gln Phe Asn Asn Leu His Val Ala Ser Ile Leu Lys Phe Cys Lys 325 330 335 Thr Glu Gly Tyr Leu Ile Val Gly Met Asp Gly Pro Asp Ala Leu Glu 340 345 350 Asp Ser Phe Pro Ile His Ile Asn Asn Thr Phe Met Phe Pro Val Gly 355 360 365 Tyr Ala Glu Lys Tyr Asn Leu Glu Leu Val Pro Pro Asp Glu Phe Lys 370 375 380 Gly Thr Phe Arg Trp Asp Glu Tyr Leu Glu Lys Glu Ser Ala Glu Thr 385 390 395 400 Leu Pro Leu Asp Leu Phe Lys Pro Met Pro Ser Gln Glu Arg Leu Asp 405 410 415 Lys Phe Lys Val Ile Leu Ile Ser Lys Arg Val Gly Leu Arg Leu Glu 420 425 430 Ala Ala Asp Met Cys Glu Asn Gln Phe Ile Cys Pro Ala Thr Val Lys 435 440 445 Ser Val His Gly Arg Leu Ile Asn Val Asn Phe Asp Gly Trp Asp Glu 450 455 460 Glu Phe Asp Glu Leu Tyr Asp Val Asp Ser His Asp Ile Leu Pro Ile 465 470 475 480 Gly Trp Cys Glu Ala His Ser Tyr Val Leu Gln Pro Pro Lys Lys Tyr 485 490 495 Asn Tyr 6 1497 DNA Caenorhabditis elegans 6 atgtctgaat ttctgaaaat tgtcagagct aacaaaaaat cggacagaaa actcgataag 60 acctacttgt gggaatccta tttacatcag ttcgagaaag gaaaaacttc tttcattcca 120 gttgaagcat tcaatcgtaa ccttacagtt aattttaacg aatgcgtgaa ggaaggagtt 180 atcttcgaaa cagtggtcca tgattatgac aagaactgcg attcgattca agtcagatgg 240 tttgcacgaa ttgaaaaagt ttgcggatac agagttctgg ctcagtttat cggagctgac 300 acgaaatttt ggctcaatat tttatcggac gatatgtttg gtttggcaaa cgccgcaatg 360 agtgatccca atatggataa aattgtatat gctccgccgc ttgcaatcaa cgaagaatac 420 caaaatgata tggtaaatta tgtaaataat tgcattgatg gcgaaatcgt cggccaaact 480 tcgctgtctc caaaattcga tgaagggaag gctctcctaa gcaagcatcg tttcaaagtt 540 ggacaacgtc ttgaactatt aaattattcc aattctactg aaatacgcgt agcgcgaatt 600 caagaaatat gtggacgacg aatgaatgta tctatcacaa agaaagactt tcccgaatcg 660 cttccagatg cagatgacga cagacaagtc tttagctctg gatctcaata ttggatagac 720 gagggaagct tcttcatatt tcctgttgga tttgcagcag tcaatggata tcaactaaat 780 gcgaaaaagg aatatattga gcacacaaat aaaattgctc aagcaataaa aaatggagaa 840 aatccaagat atgactcaga cgacgtcaca tttgatcaat tagcaaaaga tccaattgat 900 cccatgattt ggagaaaagt taaggttgga caaaagtttg agctcatcga ccccttggct 960 cagcaattca ataacctcca cgtcgcttcg attctcaaat tttgcaaaac tgaaggatat 1020 cttattgtgg gaatggatgg tccagatgca cttgaagaca gttttcctat tcatatcaat 1080 aatacattta tgttcccagt tggttatgcg gaaaagtata atttggaact tgttccgcca 1140 gatgagttca aaggaacatt cagatgggat gaatacttgg agaaagaatc tgcagaaacc 1200 ctaccgcttg acttgttcaa gccaatgcct tcccaagaga gattagacaa atttaaggta 1260 attctgattt ccaaacgggt aggactacgc cttgaagctg ctgacatgtg tgaaaatcag 1320 tttatttgtc cagctacagt gaaatcagtt catggaagac tgataaatgt caatttcgac 1380 ggctgggatg aagaatttga tgaactgtat gatgtggact cccatgatat tctaccgata 1440 ggatggtgtg aagcgcacag ttatgttcta caacctccga aaaagtacaa ctattga 1497 7 100 PRT Artificial Sequence derived from Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens 7 Phe Asp Trp Glu Asp Tyr Leu Glu Glu Thr Gly Ala Arg Ala Ala Pro 1 5 10 15 Val Glu Leu Phe Asp Lys Gln Pro Val Asp Ser Pro Pro Asn Gly Phe 20 25 30 Lys Val Gly Met Lys Leu Glu Ala Val Asp Pro Arg Asn Pro Ser Leu 35 40 45 Ile Cys Val Ala Thr Val Val Glu Val Lys Gly Tyr Arg Leu Leu Leu 50 55 60 His Phe Asp Gly Trp Asp Asp Arg Tyr Asp Phe Trp Cys Asp Ala Asp 65 70 75 80 Ser Pro Asp Ile Phe Pro Val Gly Trp Cys Glu Lys Asn Gly His Pro 85 90 95 Leu Gln Pro Pro 100 8 100 PRT Drosophila melanogaster 8 Trp Ser Trp Glu Ser Tyr Leu Glu Glu Gln Lys Ala Ile Thr Ala Pro 1 5 10 15 Val Ser Leu Phe Asp Ser Gln Ala Val Thr His Asn Lys Asn Gly Phe 20 25 30 Lys Leu Gly Met Lys Leu Glu Gly Ile Asp Pro Gln His Pro Ser Met 35 40 45 Tyr Phe Ile Leu Thr Val Ala Glu Val Cys Gly Tyr Arg Leu Arg Leu 50 55 60 His Phe Asp Gly Tyr Ser Glu Cys His Asp Phe Trp Val Asn Ala Asn 65 70 75 80 Ser Pro Asp Ile His Pro Ala Gly Trp Phe Glu Lys Thr Gly His Lys 85 90 95 Leu Gln Leu Pro 100 9 100 PRT Drosophila melanogaster 9 Phe Ser Trp Ser Gln Tyr Met Cys Ser Thr Arg Ala Gln Ala Ala Pro 1 5 10 15 Lys His Met Phe Val Ser Gln Ser His Ser Pro Pro Pro Leu Gly Phe 20 25 30 Gln Val Gly Met Lys Leu Glu Ala Val Asp Arg Met Asn Pro Ser Leu 35 40 45 Val Cys Val Ala Ser Val Thr Asp Val Val Asp Ser Arg Phe Leu Val 50 55 60 His Phe Asp Asn Trp Asp Asp Thr Tyr Asp Tyr Trp Cys Asp Pro Ser 65 70 75 80 Ser Pro Tyr Ile His Pro Val Gly Trp Cys Gln Lys Gln Gly Lys Pro 85 90 95 Leu Thr Pro Pro 100 10 96 PRT Drosophila melanogaster 10 Phe Cys Trp Glu Lys Tyr Leu Glu Glu Thr Gly Ala Ser Ala Val Pro 1 5 10 15 Thr Trp Ala Phe Lys Val Arg Pro Pro His Ser Phe Leu Val Asn Met 20 25 30 Lys Leu Glu Ala Val Asp Arg Arg Asn Pro Ala Leu Ile Arg Val Ala 35 40 45 Ser Val Glu Asp Val Glu Asp His Arg Ile Lys Ile His Phe Asp Gly 50 55 60 Trp Ser His Gly Tyr Asp Phe Trp Ile Asp Ala Asp His Pro Asp Ile 65 70 75 80 His Pro Ala Gly Trp Cys Ser Lys Thr Gly His Pro Leu Gln Pro Pro 85 90 95 11 99 PRT Drosophila melanogaster 11 Phe Arg Trp Ser Glu Tyr Leu Ser Lys Gly Lys Asp Val Ala Ala Pro 1 5 10 15 Ile His Leu Phe Leu Asn Pro Phe Pro Ile Ser Pro Asn Cys Phe Glu 20 25 30 Ile Gly Met Lys Leu Glu Ala Ile Asp Pro Glu Asn Cys Ser Leu Phe 35 40 45 Cys Val Cys Ser Ile Val Glu Val Arg Gly Tyr Arg Leu Lys Leu Ser 50 55 60 Phe Asp Gly Tyr Ser Ser Met Tyr Asp Phe Trp Val Asn Ala Asp Ser 65 70 75 80 Gln Asp Ile Phe Pro Pro Gly Trp Cys Asp Glu Thr Ala Arg Val Leu 85 90 95 Gln Ala Pro 12 100 PRT Drosophila melanogaster 12 Phe Ser Trp Ser Arg Tyr Leu Val Lys Thr Gly Gly Lys Ala Ala Pro 1 5 10 15 Arg Ala Leu Phe Asn Met Gln Gln Gln Met Asp Val Arg Asn Gly Phe 20 25 30 Ala Val Gly Met His Leu Glu Ala Glu Asp Leu Asn Asp Thr Gly Lys 35 40 45 Ile Cys Val Ala Thr Val Thr Asp Ile Leu Asp Glu Arg Ile Arg Val 50 55 60 His Phe Asp Gly Trp Asp Asp Cys Tyr Asp Leu Trp Val His Ile Thr 65 70 75 80 Ser Pro Tyr Ile His Pro Cys Gly Trp His Glu Gly Arg Gln Gln Leu 85 90 95 Ile Val Pro Pro 100 13 96 PRT Drosophila melanogaster 13 Phe Ile Trp Asp Asp Tyr Ile Ser Glu Val Gly Gly Met Ala Ala Ser 1 5 10 15 Lys Glu Leu Phe Thr Pro Arg Gln Pro Met Glu Tyr Gln Glu Arg Met 20 25 30 Lys Leu Glu Val Val Asp Gln Arg Asn Pro Cys Leu Ile Arg Pro Ala 35 40 45 Thr Val Val Thr Arg Lys Gly Tyr Arg Val Gln Leu His Leu Asp Cys 50 55 60 Trp Pro Thr Glu Tyr Tyr Phe Trp Leu Glu Asp Asp Ser Pro Asp Leu 65 70 75 80 His Pro Ile Gly Trp Cys Glu Ala Thr Ser His Glu Leu Glu Thr Pro 85 90 95 14 99 PRT Mus musculus 14 Phe Thr Trp Asp Lys Tyr Leu Lys Glu Thr Cys Ser Val Pro Ala Pro 1 5 10 15 Val His Cys Phe Lys Gln Ser Tyr Thr Pro Pro Ser Asn Glu Phe Lys 20 25 30 Ile Ser Met Lys Leu Glu Ala Gln Asp Pro Arg Asn Thr Thr Ser Thr 35 40 45 Cys Ile Ala Thr Val Val Gly Leu Thr Gly Ala Arg Leu Arg Leu Arg 50 55 60 Leu Asp Gly Ser Asp Asn Lys Asn Asp Phe Trp Arg Leu Val Asp Ser 65 70 75 80 Ser Glu Ile Gln Pro Ile Gly Asn Cys Glu Lys Asn Gly Gly Met Leu 85 90 95 Gln Pro Pro 15 100 PRT Homo sapiens 15 Ser Ser Trp Pro Met Phe Leu Thr Leu Asn Gly Ser Glu Met Ala Ser 1 5 10 15 Ala Thr Leu Phe Lys Lys Glu Pro Pro Lys Pro Pro Leu Asn Asn Phe 20 25 30 Lys Val Gly Met Lys Leu Glu Ala Ile Asp Lys Lys Asn Pro Tyr Leu 35 40 45 Ile Cys Pro Ala Thr Ile Gly Asp Val Lys Gly Asp Glu Val His Ile 50 55 60 Thr Phe Asp Gly Trp Ser Gly Ala Phe Asp Tyr Trp Cys Lys Tyr Asp 65 70 75 80 Ser Arg Asp Ile Phe Pro Ala Gly Trp Cys Arg Leu Thr Gly Asp Val 85 90 95 Leu Gln Pro Pro 100 16 1276 DNA Caenorhabditis elegans 16 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ccgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 17 386 PRT Caenorhabditis elegans 17 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Pro Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 18 1276 DNA Caenorhabditis elegans 18 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtgatgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 19 386 PRT Caenorhabditis elegans 19 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Met Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 20 1276 DNA Caenorhabditis elegans 20 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttag agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 21 78 PRT Caenorhabditis elegans 21 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile 65 70 75 22 1276 DNA Caenorhabditis elegans 22 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaat aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 23 112 PRT Caenorhabditis elegans 23 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 24 1276 DNA Caenorhabditis elegans 24 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcacgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 25 386 PRT Caenorhabditis elegans 25 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu His Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 26 1276 DNA Caenorhabditis elegans 26 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggca ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 27 386 PRT Caenorhabditis elegans 27 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp His Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 28 1276 DNA Caenorhabditis elegans 28 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgagagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 29 146 PRT Caenorhabditis elegans 29 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg 145 30 1276 DNA Caenorhabditis elegans 30 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctggaagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 31 386 PRT Caenorhabditis elegans 31 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Lys Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 32 1276 DNA Caenorhabditis elegans 32 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta tttgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 33 386 PRT Caenorhabditis elegans 33 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Cys Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 34 1276 DNA Caenorhabditis elegans 34 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct aggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 35 162 PRT Caenorhabditis elegans 35 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg 36 1276 DNA Caenorhabditis elegans 36 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct ggaaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 37 386 PRT Caenorhabditis elegans 37 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Lys Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Glu Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 38 1275 DNA Caenorhabditis elegans 38 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaagtgg attctgggga gctcatggag 600 cccatggagc ccatggattc tacaatggat gagatgtgcg tcgaggagga gccctacgag 660 gagacagggt ccaattggag cgatccggcg ccggaaccat cccaatccaa atcccagtcc 720 ccagaagcca agtaccctca agcctaccta ctacctgagg cggacgaagt ctacaatcct 780 gacgatttct atcaagagga acatgaatcc gcatcaaacg ccatgtatcg gatcgctttc 840 tcacagcagt acggtggcgg cgggtcccca gccgtgcaga agcccgtcac ttttagtgct 900 cagccggcgc cggcgccagt tagagaggcc ccaagcccag ttgtggagaa tgttagttca 960 tcgagtttca ccccgaagcc cccggccatg atcaacaatt ttggtgagga gatgaaccaa 1020 ataacatacc aagcgatccg tattgcccga gagcagccgg aacgtctgaa attgctccgt 1080 aaggcacttt tcgacgttgt cctggcgttt gatcagaagg aatacgccga tgttggggat 1140 ttgtacaggg atttggcgca aaagaattcg tgataatttt tttttgagtt ttttaatttt 1200 taatttattt caatttttgt tacatgttcc aatataataa acaggtgctt gtttaaaaaa 1260 aaaaaaaaaa aaaaa 1275 39 325 PRT Caenorhabditis elegans 39 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Trp Ile Leu Gly 180 185 190 Ser Ser Trp Ser Pro Trp Ser Pro Trp Ile Leu Gln Trp Met Arg Cys 195 200 205 Ala Ser Arg Arg Ser Pro Thr Arg Arg Gln Gly Pro Ile Gly Ala Ile 210 215 220 Arg Arg Arg Asn His Pro Asn Pro Asn Pro Ser Pro Gln Lys Pro Ser 225 230 235 240 Thr Leu Lys Pro Thr Tyr Tyr Leu Arg Arg Thr Lys Ser Thr Ile Leu 245 250 255 Thr Ile Ser Ile Lys Arg Asn Met Asn Pro His Gln Thr Pro Cys Ile 260 265 270 Gly Ser Leu Ser His Ser Ser Thr Val Ala Ala Gly Pro Gln Pro Cys 275 280 285 Arg Ser Pro Ser Leu Leu Val Leu Ser Arg Arg Arg Arg Gln Leu Glu 290 295 300 Arg Pro Gln Ala Gln Leu Trp Arg Met Leu Val His Arg Val Ser Pro 305 310 315 320 Arg Ser Pro Arg Pro 325 40 1276 DNA Caenorhabditis elegans 40 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagtag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 41 278 PRT Caenorhabditis elegans 41 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln 275 42 1276 DNA Caenorhabditis elegans 42 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag tttgagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 43 303 PRT Caenorhabditis elegans 43 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val 290 295 300 44 1276 DNA Caenorhabditis elegans 44 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac taagcgatcc gtattgcccg agagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 45 339 PRT Caenorhabditis elegans 45 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr 46 1276 DNA Caenorhabditis elegans 46 agaatctgcc aaaatgtcaa agataaagac acattccact ggctcaaaac ggacggtacc 60 attctacaag ctgccaccgc ccgtgccact tccaccactc ccgccacccg atccaacccg 120 gtacttctcg acggaaaagt acatcgcact gagcaaagat gagaaattca aatttgatga 180 ttacgatgtg aatgatgaga cgctgaaaaa agtggtgctc aacgagattg gcaagtgccc 240 ggatatttgg agctcgcgga gccaggcagc cattatggag cactatccga ttgttgcaac 300 tgaaacgtac aggaggacag ggttgctgtt gtctatcaaa tcgctgaaac aaatctacaa 360 atgcggaaag gacaatctcc gaaaccggct tcgcgtggca attgtaagca agcggcttac 420 accggcccaa gtagaggcct atatgtggcg ctgggagttt tacggcttta ttcgctacta 480 tcgagactat acacaacgct gggaggccga cttgttgaaa gatttggacg tggtgctcgg 540 gctcgaggct cggcgagcat cgaaaaatat ggaaaaggtg gattctgggg agctcatgga 600 gcccatggag cccatggatt ctacaatgga tgagatgtgc gtcgaggagg agccctacga 660 ggagacaggg tccaattgga gcgatccggc gccggaacca tcccaatcca aatcccagtc 720 cccagaagcc aagtaccctc aagcctacct actacctgag gcggacgaag tctacaatcc 780 tgacgatttc tatcaagagg aacatgaatc cgcatcaaac gccatgtatc ggatcgcttt 840 ctcacagcag tacggtggcg gcgggtcccc agccgtgcag aagcccgtca cttttagtgc 900 tcagccggcg ccggcgccag ttagagaggc cccaagccca gttgtggaga atgttagttc 960 atcgagtttc accccgaagc ccccggccat gatcaacaat tttggtgagg agatgaacca 1020 aataacatac caagcgatcc gtattgcccg aaagcagccg gaacgtctga aattgctccg 1080 taaggcactt ttcgacgttg tcctggcgtt tgatcagaag gaatacgccg atgttgggga 1140 tttgtacagg gatttggcgc aaaagaattc gtgataattt ttttttgagt tttttaattt 1200 ttaatttatt tcaatttttg ttacatgttc caatataata aacaggtgct tgtttaaaaa 1260 aaaaaaaaaa aaaaaa 1276 47 386 PRT Caenorhabditis elegans 47 Met Ser Lys Ile Lys Thr His Ser Thr Gly Ser Lys Arg Thr Val Pro 1 5 10 15 Phe Tyr Lys Leu Pro Pro Pro Val Pro Leu Pro Pro Leu Pro Pro Pro 20 25 30 Asp Pro Thr Arg Tyr Phe Ser Thr Glu Lys Tyr Ile Ala Leu Ser Lys 35 40 45 Asp Glu Lys Phe Lys Phe Asp Asp Tyr Asp Val Asn Asp Glu Thr Leu 50 55 60 Lys Lys Val Val Leu Asn Glu Ile Gly Lys Cys Pro Asp Ile Trp Ser 65 70 75 80 Ser Arg Ser Gln Ala Ala Ile Met Glu His Tyr Pro Ile Val Ala Thr 85 90 95 Glu Thr Tyr Arg Arg Thr Gly Leu Leu Leu Ser Ile Lys Ser Leu Lys 100 105 110 Gln Ile Tyr Lys Cys Gly Lys Asp Asn Leu Arg Asn Arg Leu Arg Val 115 120 125 Ala Ile Val Ser Lys Arg Leu Thr Pro Ala Gln Val Glu Ala Tyr Met 130 135 140 Trp Arg Trp Glu Phe Tyr Gly Phe Ile Arg Tyr Tyr Arg Asp Tyr Thr 145 150 155 160 Gln Arg Trp Glu Ala Asp Leu Leu Lys Asp Leu Asp Val Val Leu Gly 165 170 175 Leu Glu Ala Arg Arg Ala Ser Lys Asn Met Glu Lys Val Asp Ser Gly 180 185 190 Glu Leu Met Glu Pro Met Glu Pro Met Asp Ser Thr Met Asp Glu Met 195 200 205 Cys Val Glu Glu Glu Pro Tyr Glu Glu Thr Gly Ser Asn Trp Ser Asp 210 215 220 Pro Ala Pro Glu Pro Ser Gln Ser Lys Ser Gln Ser Pro Glu Ala Lys 225 230 235 240 Tyr Pro Gln Ala Tyr Leu Leu Pro Glu Ala Asp Glu Val Tyr Asn Pro 245 250 255 Asp Asp Phe Tyr Gln Glu Glu His Glu Ser Ala Ser Asn Ala Met Tyr 260 265 270 Arg Ile Ala Phe Ser Gln Gln Tyr Gly Gly Gly Gly Ser Pro Ala Val 275 280 285 Gln Lys Pro Val Thr Phe Ser Ala Gln Pro Ala Pro Ala Pro Val Arg 290 295 300 Glu Ala Pro Ser Pro Val Val Glu Asn Val Ser Ser Ser Ser Phe Thr 305 310 315 320 Pro Lys Pro Pro Ala Met Ile Asn Asn Phe Gly Glu Glu Met Asn Gln 325 330 335 Ile Thr Tyr Gln Ala Ile Arg Ile Ala Arg Lys Gln Pro Glu Arg Leu 340 345 350 Lys Leu Leu Arg Lys Ala Leu Phe Asp Val Val Leu Ala Phe Asp Gln 355 360 365 Lys Glu Tyr Ala Asp Val Gly Asp Leu Tyr Arg Asp Leu Ala Gln Lys 370 375 380 Asn Ser 385 48 1108 DNA Caenorhabditis elegans 48 gcaaaaaact agatattttg tggcattttt acaattaaaa aacctttaaa aaatggatca 60 ccatgctatg taccgaaccg ctgaattcaa caaaactact gtccgattat tggcggaatt 120 catcgaaaag actgggcaga atgcgacgat agtgaatatg gacagctttt tggagttctt 180 tgcgtatttg aatcccacgg ctccaattcc aacggttcca gaaattgaaa aataattatt 240 gctaaaatca ccgattcgtt gcattgtgtg tggaatggaa actgaatcag attccgcagt 300 gacattaagc atcgataatg cttcaattat tctcacagcg acagtaattg gttactgtag 360 agatccaagt gatgcagtta atcaaattcg aaaggagagt cttcgagcat gcacgaaaca 420 tttcaacagt attttccatg tcatcttcga aggactgcaa atcgagaaca cctactgtgc 480 tcatcatgca aaatacagtc ttgccaatcg ttggtgcaaa gtctacacga tgattcgatc 540 ttccctgggc gagcagttca caaagttcga tgtgcgcaat tttaaatcaa tattgcaatc 600 atttttggat acttttggtg aaattgatga cgacaaaaag gataaagaat cttctcattt 660 tgatgaatgt tttgaagaaa tggattcaga aaacgtagaa attaaaatgg agagcccaca 720 agaagaagct gcagagaaat cgaagttttc tgaaaaccta gtggaggtaa aactggaacc 780 aattgaaact catgaacttg acaaaactat atccgacttt tcttcaagtg atataattga 840 ttcgtcccaa aaactgcagc aaaatggttt tcctgaaaaa gtggagcaaa tggacaaata 900 tagcaacaaa ttgaaagatg aagcttcaga caaaaagtat gaaaagccag gaaaaaagga 960 ctacgttgaa gaagagggat actgggcgcc gatcaccgac agcgaggatg atgaagcctg 1020 aatttattta atcaaacgtt ttggaaattt tttttgtttt tgtcaataaa accatataac 1080 aataaaaaaa aaaaaaaaaa aactcgag 1108 49 60 PRT Caenorhabditis elegans 49 Met Asp His His Ala Met Tyr Arg Thr Ala Glu Phe Asn Lys Thr Thr 1 5 10 15 Val Arg Leu Leu Ala Glu Phe Ile Glu Lys Thr Gly Gln Asn Ala Thr 20 25 30 Ile Val Asn Met Asp Ser Phe Leu Glu Phe Phe Ala Tyr Leu Asn Pro 35 40 45 Thr Ala Pro Ile Pro Thr Val Pro Glu Ile Glu Lys 50 55 60 50 50 000 51 60 PRT Caenorhabditis elegans 51 Arg Cys Ile Val Cys Gly Met Glu Thr Glu Ser Asp Ser Ala Val Thr 1 5 10 15 Leu Ser Ile Asp Asn Ala Ser Ile Ile Leu Thr Ala Thr Val Ile Gly 20 25 30 Tyr Cys Arg Asp Pro Ser Asp Ala Val Asn Gln Ile Arg Lys Glu Ser 35 40 45 Leu Arg Ala Cys Thr Lys His Phe Asn Ser Ile Phe 50 55 60 52 60 PRT Caenorhabditis elegans 52 Pro Cys Ile Leu Cys Glu Lys Ala Leu Leu Met Arg Glu Ser Ile Ala 1 5 10 15 Met Thr Asp Asn Glu Ala Val Lys Val Leu Met Ala Ala Val Met Ser 20 25 30 Gly His Phe Arg Met Ala Thr Ala Glu Lys Ala Ile Arg His Glu Arg 35 40 45 Leu Arg Met Cys Tyr Asp His Val Asp Phe Val Tyr 50 55 60 53 60 PRT Caenorhabditis elegans 53 Pro Cys Ile Ile Cys Gly Asn Glu Val Pro Gly His Arg Ser Ile Arg 1 5 10 15 Val Ser Asp Asp Asp Ala Ala Ile Phe Leu Thr Ala Ala Val Leu Thr 20 25 30 Asp Gln Lys Thr Ile Arg Gln Ala Lys Arg Asp Ile Leu Ser Glu Tyr 35 40 45 Leu Thr Val Cys Leu Arg His Ser Leu His Tyr Tyr 50 55 60 54 60 PRT Caenorhabditis elegans 54 Pro Cys Leu Val Cys Asn Gln Gln Met Glu Met Thr Lys Val Arg Ser 1 5 10 15 Val Asn Asn Thr Asp Ala Tyr Ile Met Ile Tyr Val Cys Val Met Asn 20 25 30 Asp Lys Tyr Asp Met Asp Lys Ala Lys Glu Leu Ala Arg Met Gln Arg 35 40 45 Phe Lys Cys Cys Val Ser His Leu Asp Glu Leu Tyr 50 55 60 55 177 PRT Caenorhabditis elegans 55 Met Leu Ser Ile Lys Gln Glu Leu Leu Asp Ala Pro Pro Pro Pro Pro 1 5 10 15 Ala Ala Thr Pro Leu Pro Pro Ile Thr His Arg Ile Ser Leu Ser Gly 20 25 30 Tyr Arg Asn Ile His Ala Lys Ser Phe Leu Lys Thr Met Thr Met Asp 35 40 45 Leu Cys Val Arg Arg Val Val Leu Ser Leu Leu Glu Asn Arg Arg Ala 50 55 60 Leu Trp Ile Arg Val His Lys Ser Pro Lys Ala Asp Trp Glu Val Leu 65 70 75 80 Gly Val Glu Val Phe Glu Arg Thr Gly Lys Ala Val Ser Val Lys Gln 85 90 95 Leu Gln Arg Ile Phe Leu Thr Ala Arg Asp Trp Leu Arg Arg Asn Leu 100 105 110 Gln Leu Tyr Ile Ile Gln Arg Lys Met Asp Lys Leu Thr Leu Asp Ala 115 120 125 Glu Leu Ala Lys Trp Glu Leu Tyr Pro His Phe Ile Tyr Tyr Arg Gln 130 135 140 Tyr Leu Gly Gln Phe Glu Ala His Leu Arg Gly Glu Glu Trp Thr Gly 145 150 155 160 Glu Leu Tyr Asp Asp Asp Ile Ile Cys Asp Gly Ile Met Gln Val Glu 165 170 175 Val 56 75 PRT Caenorhabditis elegans 56 Glu Asp Ser Val Ser Tyr Thr Lys Ile Thr Glu Asp Leu Leu Gln Lys 1 5 10 15 Lys Pro His Lys His Arg Phe Ile Arg Gln Ala Leu Phe Lys Thr Ile 20 25 30 Met Ala Leu Asp Asp Asp Glu Val Glu Tyr Thr Glu Leu Ala Asp Leu 35 40 45 Phe Gly Asp Ile Ala Glu Gln Ser Asn Val Val Arg Arg Leu Arg Leu 50 55 60 Gln Arg Gln Gln Gln Arg Gly Arg Gly Glu Gln 65 70 75 57 178 PRT Caenorhabditis elegans 57 Met Ser Leu Ile Lys Gln Glu His Met His Pro Pro Pro Arg Ala Ile 1 5 10 15 Thr Pro Leu Pro Pro Ala Thr His Gln Ile Thr Leu Glu Glu Tyr Lys 20 25 30 Glu Arg Glu Lys Lys Asp Tyr Tyr Arg Asp Ala Thr Lys Asp Ala Ser 35 40 45 Val Lys Lys Val Val Leu Ser Leu Leu Lys Asp His Pro Gly Met Trp 50 55 60 Gln Asn Gly Asn Arg Phe Gln Pro Glu Lys Trp Arg Ala Leu Gly Val 65 70 75 80 Asp Val Tyr Gln Arg Thr Gly Gln Ile Val Arg Val Asn Asp Met Arg 85 90 95 Lys Met Leu Val Met Gly Lys Ser Val Leu Lys Lys Lys Ile Ala Ile 100 105 110 Cys Ile Arg Asp Lys Lys Leu Asp Arg Ala Ala Thr Glu Lys Asp Leu 115 120 125 Trp Tyr Trp Glu Tyr Tyr Arg His Phe Leu Tyr Tyr Arg Glu Thr Leu 130 135 140 Gly Gln Phe Glu Ala Asn Leu Arg Gly Glu Glu Trp Thr Gly Glu Asp 145 150 155 160 Gln Ile Gln Asp Glu Asp Asp Ile Ile Tyr Asp Gly Met Leu Asp Gly 165 170 175 Asp Leu 58 73 PRT Caenorhabditis elegans 58 Arg Ser Ala Gln His Ile Ala Glu Gln Ala Lys Arg Leu Phe Leu Gln 1 5 10 15 Tyr Pro Glu Lys Ser Asn Leu Ile Arg Glu Thr Met Phe Lys Thr Ile 20 25 30 Leu Ala Phe Asp Asp Pro Ser Ala Asp Tyr Gln Asn Val Gly Glu Ile 35 40 45 Phe Asp Asp Leu Ala Ala Gln Glu Ala Ala Lys Lys Arg Lys Arg Ala 50 55 60 Glu Asn Arg Ala Gln Arg Glu Gln Gln 65 70 59 179 PRT Caenorhabditis elegans 59 Met Ser Leu Ile Lys Gln Glu His Met Asn Pro Pro Pro Arg Thr Ile 1 5 10 15 Thr Pro Leu Pro Pro Pro Thr His Gln Ile Thr Ile Glu Glu Tyr Lys 20 25 30 Glu Arg Val Lys Arg Asp Tyr Tyr Arg Asn Ala Thr Lys Asp Thr Ser 35 40 45 Leu Lys Lys Val Val Leu Ser Leu Ile Lys Asp Arg Lys Ala Met Trp 50 55 60 Ala Pro Ala Ala Lys Pro Ser Glu Asp Lys Trp Gln Lys Leu Gly Ala 65 70 75 80 Glu Val Phe Ser Arg Thr Gly Lys Val Val Ser Val Thr Gln Leu Arg 85 90 95 Arg Met Leu Val Ser Ser Lys His Val Leu Lys Thr Lys Met Ser His 100 105 110 Cys Ile Lys Val Lys Lys Met Asp Arg Val Ser Thr Glu Ala Tyr Leu 115 120 125 Trp Asn Trp Glu Phe Tyr Arg His Phe Leu Tyr Tyr Arg Glu Met Leu 130 135 140 Asp Arg Phe Glu Ala Asn Leu Arg Gly Lys Gln Trp Thr Gly Glu Asp 145 150 155 160 Gln Pro Thr Asp Asp Asp Asp Asp Ile Ile Cys Asp Gly Ile Phe Glu 165 170 175 Val Glu Met 60 70 PRT Caenorhabditis elegans 60 Ser Thr Ala Glu Gln Ile Gly Glu Glu Ile Asp Arg Leu Ile Gln Leu 1 5 10 15 Tyr Pro Gln Arg Glu Met Leu Ile Arg Gln Ala Phe Phe Lys Thr Ile 20 25 30 Phe Ala Leu Glu Asp Glu Thr Val Glu Phe Ser Asn Leu Gly Asp Leu 35 40 45 Phe Glu Asp Leu Ala Glu Gln Glu Asn Phe Lys Arg Arg Arg Arg Ser 50 55 60 Arg Ala Gln Arg Leu Glu 65 70 61 187 PRT Caenorhabditis elegans 61 Met Leu Asn Ile Lys Gln Glu Gly Val Val Ala Asp Ala Pro Arg Ala 1 5 10 15 Leu Thr Pro Ile Pro Pro Phe Ile His His Val Ser Met Glu Glu Tyr 20 25 30 Met Gly Met Glu Leu Asn Ser Val Tyr Glu Glu Ala Thr Lys Asp Ser 35 40 45 Ala Leu Lys Lys Val Val Leu Asp Leu Leu Lys Asp Arg Pro Gly Met 50 55 60 Trp Gln Asn Gly Asn Arg Phe Gln Leu Glu Asn Trp Arg Glu Leu Gly 65 70 75 80 Val Asp Val Tyr Gln Arg Thr Gly Gln Ile Val Arg Ala Glu Leu Gly 85 90 95 Glu Val Ser Val Asn Asp Met His Arg Met Phe Val Val Gly Lys Ala 100 105 110 Val Leu Lys Gln Lys Ile Thr Val Cys Ile Arg Tyr Lys Lys Leu Asp 115 120 125 Arg Ala Ala Thr Glu Ala Asp Leu Gln Asn Trp Glu Phe Tyr Arg His 130 135 140 Phe Arg Tyr Tyr Arg Glu Thr Leu Gly Gln Phe Glu Ala Asn Leu Arg 145 150 155 160 Gly Glu Gln Trp Thr Gly Glu Asp Gln Pro Ala Asp Asp Asp Asp Asp 165 170 175 Ile Ile Tyr Asp Gly Ile Phe Glu Val Glu Met 180 185 62 69 PRT Caenorhabditis elegans 62 Ser Thr Ala Glu Gln Ile Gly Glu Glu Ile Asp Arg Leu Ile Gln Leu 1 5 10 15 Tyr Pro Gln Arg Glu Met Leu Ile Arg Gln Ala Phe Phe Lys Thr Ile 20 25 30 Phe Ala Leu Glu Asp Glu Thr Val Glu Phe Ser Asn Leu Gly Asp Leu 35 40 45 Phe Glu Asp Leu Ala Glu Gln Glu Asn Phe Lys Arg Arg Arg Arg Ser 50 55 60 Ala Gln Arg Leu Glu 65 63 186 PRT Caenorhabditis elegans 63 Met Met Asn Pro Lys Glu Glu Pro Arg Pro Phe Ser Ile Val Pro Leu 1 5 10 15 Pro Arg Pro Pro Arg Pro Thr Thr Pro Leu Pro Pro Ile Ser His Cys 20 25 30 Ile Thr Met Ala Asp Tyr Leu Leu Leu Glu Asn Thr Lys Phe His Lys 35 40 45 Thr Ala Thr Arg Ala Pro Lys Ile Lys Lys Val Leu Leu Ser Leu Leu 50 55 60 Lys Asp Arg Pro Glu Ile Trp Asp Arg Lys Ala Gln Phe Ser Ala Lys 65 70 75 80 Asn Trp Gln Asn Leu Gly Val Glu Val Tyr Glu Arg Thr Gly Tyr Ile 85 90 95 Val Arg Ser Asn Asp Leu His Lys Met Leu Arg Thr Ala Lys Val Val 100 105 110 Leu Lys Asn Lys Leu Arg Thr Cys Ile Gly Ile Lys Lys Leu Asp Arg 115 120 125 Ala Ala Thr Glu Thr Glu Leu Trp Lys Trp Glu Tyr Tyr Pro His Phe 130 135 140 Ile Tyr Tyr Arg Glu Thr Leu Gly His Phe Glu Ala Asn Leu Arg Gly 145 150 155 160 Glu Pro Trp Asp Gly Glu Ala His Ile Asp Asp Asp Asp Asp Asp Ile 165 170 175 Ile Tyr Glu Gly Tyr Trp Glu Ala Asp Lys 180 185 64 70 PRT Caenorhabditis elegans 64 Asn Ser Ala Gln His Ile Gly Glu Gln Val His Arg Leu Phe Ala Gln 1 5 10 15 Tyr Pro Glu Arg Ser Lys Leu Phe Arg Glu Thr Leu Phe Lys Thr Ile 20 25 30 Leu Ala Leu Glu Glu Pro Glu Tyr Glu His Ala Ala Glu Val Phe Thr 35 40 45 Asp Leu Ala Gln Ser Glu Thr Ala Lys Arg Arg Arg Arg Ser Glu Ala 50 55 60 Thr Trp Gln Asn Gly Gln 65 70 65 186 PRT Caenorhabditis elegans 65 Met Val Ser Ala Thr Arg Val Pro Arg Arg Ser Ser Thr Thr Thr Ser 1 5 10 15 Ala Thr Ala Gln Gln Arg Thr Pro Ser Pro Leu Met Pro Ala Ser Phe 20 25 30 Pro Ile Thr Met Asp Glu Tyr Leu Glu Lys Glu Asn Arg Glu Phe Val 35 40 45 Val Asn Ala Ser Lys Asp Ile Ala Met Lys Lys Leu Ala Leu Thr Leu 50 55 60 Leu Glu Leu Tyr Pro Glu Met Trp Lys Pro Gly Gly Pro Met Val Ala 65 70 75 80 Lys Lys Trp Gln Ala Phe Gly Ala Glu Met Tyr Arg Arg Thr Gly Lys 85 90 95 Ile Tyr Arg Cys Lys Asp Leu His Ser Val Phe Thr Leu Thr Lys Ser 100 105 110 Ser Ile Lys Arg Lys Leu Arg Thr Cys Ile Leu Ile Lys Arg Met His 115 120 125 Arg Ser Lys Thr Asp Glu Glu Met Trp Lys Tyr Glu Leu Tyr Pro Tyr 130 135 140 Phe Gln Tyr Tyr Arg Gln Ser Ile Gly Gln Phe Glu Ala Lys Leu Arg 145 150 155 160 Asp Glu Pro Trp Thr Gly Glu Asp Gln Ala Gln Glu Asp Asp Asp Ile 165 170 175 Leu Phe Asp Gly Leu Phe Glu Val Glu Asn 180 185 66 66 PRT Caenorhabditis elegans 66 Lys Thr Ala Asp Asn Ile Gly Asp Gln Val Lys Gln Leu Phe Val Asp 1 5 10 15 His Pro Asp Arg Ala Asn Phe Phe Arg Glu Val Leu Phe Lys Thr Val 20 25 30 Leu Glu Leu Arg Asp Pro Ala Phe Thr Asn Ala Gly Val Phe Phe Asp 35 40 45 Glu Met Ser Ser Leu Glu Ser Ala Lys Arg Arg Arg Arg Ser Glu Met 50 55 60 Asn Lys 65 67 178 PRT Caenorhabditis elegans 67 Met Ser Arg Ile Lys Gln Glu Gln Val Asn Pro Pro Pro Pro Pro Arg 1 5 10 15 Ala Ile Thr Pro Leu Pro Pro Ala Thr His Arg Ile Thr Met Asp Glu 20 25 30 Tyr Lys Lys Arg Glu Lys Lys Asp Tyr Tyr Arg Asp Ala Thr Lys Asp 35 40 45 Ala Ser Val Lys Lys Val Val Leu Ser Leu Leu Lys Asp Tyr Pro Asp 50 55 60 Met Trp Gln Asn Gly Asn Arg Phe Gln Thr Arg Lys Trp Arg Ala Leu 65 70 75 80 Gly Val Glu Val Tyr Gln Arg Thr Gly Gln Ile Val Gly Val Asp Asp 85 90 95 Met Arg Lys Met Phe Met Ser Gly Lys Thr Val Leu Lys Gln Lys Ile 100 105 110 Thr Phe Cys Ile Arg Asn Met Lys Met Asp Arg Ala Ala Thr Glu Ala 115 120 125 Asp Leu Gln Asn Trp Glu Tyr Tyr Arg His Phe Leu Tyr Tyr Arg Gln 130 135 140 Thr Leu Gly Lys Phe Glu Ala Lys Leu Arg Gly Glu Gln Trp Ile Gly 145 150 155 160 Glu Asp Gln Val Glu Asp Asp Asp Glu Asp Asp Val Ile Phe Asp Gly 165 170 175 Glu Ser 68 410 PRT Caenorhabditis elegans 68 Met Gly Thr Cys Trp Gly Asp Ile Ser Glu Asn Val Arg Val Glu Val 1 5 10 15 Pro Asn Thr Asp Cys Ser Leu Pro Thr Lys Val Phe Trp Ile Ala Gly 20 25 30 Ile Val Lys Leu Ala Gly Tyr Asn Ala Leu Leu Arg Tyr Glu Gly Phe 35 40 45 Glu Asn Asp Ser Gly Leu Asp Phe Trp Cys Asn Ile Cys Gly Ser Asp 50 55 60 Ile His Pro Val Gly Trp Cys Ala Ala Ser Gly Lys Pro Leu Val Pro 65 70 75 80 Pro Arg Thr Ile Gln His Lys Tyr Thr Asn Trp Lys Ala Phe Leu Val 85 90 95 Lys Arg Leu Thr Gly Ala Lys Thr Leu Pro Pro Asp Phe Ser Gln Lys 100 105 110 Val Ser Glu Ser Met Gln Tyr Pro Phe Lys Pro Cys Met Arg Val Glu 115 120 125 Val Val Asp Lys Arg His Leu Cys Arg Thr Arg Val Ala Val Val Glu 130 135 140 Ser Val Ile Gly Gly Arg Leu Arg Leu Val Tyr Glu Glu Ser Glu Asp 145 150 155 160 Arg Thr Asp Asp Phe Trp Cys His Met His Ser Pro Leu Ile His His 165 170 175 Ile Gly Trp Ser Arg Ser Ile Gly His Arg Phe Lys Arg Ser Asp Ile 180 185 190 Thr Lys Lys Gln Asp Gly His Phe Thr Asp Pro Pro His Leu Phe Ala 195 200 205 Lys Val Lys Glu Val Asp Gln Ser Gly Glu Trp Phe Lys Glu Gly Met 210 215 220 Lys Leu Glu Ala Ile Asp Pro Leu Asn Leu Ser Thr Ile Cys Val Ala 225 230 235 240 Thr Ile Lys Arg Val Leu Ala Asp Gly Phe Leu Met Ile Gly Ile Asp 245 250 255 Gly Ser Glu Ala Ala Asp Gly Ser Asp Trp Phe Cys Tyr His Ala Thr 260 265 270 Ser Pro Ser Ile Phe Pro Val Gly Phe Cys Glu Ile Asn Met Ile Glu 275 280 285 Leu Thr Pro Pro Arg Gly Tyr Thr Lys Leu Pro Phe Lys Trp Phe Asp 290 295 300 Tyr Leu Arg Glu Thr Gly Ser Ile Ala Ala Pro Val Lys Leu Phe Asn 305 310 315 320 Lys Asp Val Pro Asn His Gly Phe Arg Val Gly Met Lys Leu Glu Ala 325 330 335 Val Asp Leu Met Glu Pro Arg Leu Ile Cys Val Ala Thr Val Thr Arg 340 345 350 Ile Ile His Arg Leu Leu Arg Ile His Phe Asp Gly Trp Glu Glu Glu 355 360 365 Tyr Asp Gln Trp Val Asp Cys Glu Ser Pro Asp Leu Tyr Pro Val Gly 370 375 380 Trp Cys Gln Leu Thr Gly Tyr Gln Leu Gln Pro Pro Ala Ser Gln Cys 385 390 395 400 Lys Leu Val Tyr Arg Lys Gly Val Leu Leu 405 410 69 512 PRT Caenorhabditis elegans 69 Met Asn Phe Ser Asn Lys Lys Val Ile Leu Lys Ala Phe Leu Ser Lys 1 5 10 15 Asn Ile Ile Tyr Tyr Phe Gln Arg Gln Tyr Asn Tyr Lys Leu Glu Glu 20 25 30 Ala Glu Tyr Arg Tyr Phe Thr Glu Glu Arg Leu Phe Tyr Arg Arg Arg 35 40 45 Asn Pro Val Glu Lys Ile Ala Gln Arg Ile Pro Lys Pro Gln Ile Glu 50 55 60 Gly Thr Phe Thr Trp Ser Asp Glu Leu Arg Cys Asn Tyr Asp Gly Asn 65 70 75 80 Thr Gln Phe Leu Pro Val Glu Ala Leu Glu Gly Cys Leu Pro Leu Glu 85 90 95 Lys Leu Asn Gln His Leu Lys Pro Gly Phe Arg Leu Glu Val Val Val 100 105 110 Arg Pro Ser Leu Asp Pro Ser Ile Thr Thr Lys Ser Pro Glu Ile Arg 115 120 125 Trp Phe Gly Glu Val Thr Ala Val Cys Gly Phe Tyr Val Ala Ile Lys 130 135 140 Phe Val Gly Glu Leu Asn Arg Arg Pro Cys Trp Phe His Met Leu Ser 145 150 155 160 Glu Asp Ile Phe Asp Ile Gly Ser Gly Leu Lys Gln Asp Pro Ala Met 165 170 175 Lys Trp Leu Gln Tyr Arg Pro Leu Ser Leu Leu Lys Pro Met Gln Cys 180 185 190 Pro Lys Phe Trp Arg Arg Gly Ser Thr Pro Ala Pro Pro Val Pro Arg 195 200 205 Pro Thr Glu Glu Ile Leu Asp Glu Phe Gln Ala Glu Leu His Glu Asn 210 215 220 Arg Ile Ser Glu Pro Lys Ile Phe Asp Gln Leu Arg His Leu Ala His 225 230 235 240 Arg Pro Ser Arg Phe Arg Leu Asn Gln Arg Val Glu Leu Leu Asn Tyr 245 250 255 Leu Glu Pro Thr Glu Ile Arg Val Ala Arg Ile Leu Arg Ile Leu Gly 260 265 270 Arg Arg Leu Met Val Met Val Thr Ala Gln Asp Tyr Pro Glu Asp Leu 275 280 285 Pro Ser Val Glu Ala Lys Asp Arg Gln Val Gln His Glu Asn Val Glu 290 295 300 Phe Trp Val Asp Glu Ser Ser Phe Phe Leu Phe Pro Val Gly Phe Ala 305 310 315 320 Met Ile Asn Gly Leu Arg Thr Lys Ala Thr Glu Gly Tyr Leu Glu His 325 330 335 Ser Arg Arg Ile Ala Glu Gly Ser Gly Thr Glu Lys Leu Asn Leu Leu 340 345 350 Lys Val Gly Gln Lys Phe Glu Leu Leu Asp Pro Leu Ser Asp Leu Arg 355 360 365 Gln Ser Phe Cys Val Ala Thr Ile Arg Lys Ile Cys Lys Thr Pro Gly 370 375 380 Phe Leu Ile Ile Ser Pro Asp Glu Thr Glu Ser Asp Asp Glu Ser Phe 385 390 395 400 Pro Ile His Ile Asp Asn His Phe Met His Pro Val Gly Tyr Ala Glu 405 410 415 Lys Phe Gly Ile Lys Leu Asp Arg Leu Ala Gly Thr Glu Pro Gly Lys 420 425 430 Phe Lys Trp Glu Gly Tyr Leu Lys Glu Lys Gln Ala Glu Lys Ile Pro 435 440 445 Asp Glu Met Leu Arg Pro Leu Pro Ser Lys Glu Arg Arg His Met Phe 450 455 460 Glu Phe Gly Arg Val Leu Glu Ala Val Gly Gln Asn Glu Thr Tyr Trp 465 470 475 480 Ile Ser Pro Ala Ser Val Glu Glu Val His Gly Arg Thr Val Leu Ile 485 490 495 Glu Phe Gln Gly Trp Asp Ser Glu Phe Ser Glu Leu Tyr Asp Met Glu 500 505 510 70 411 PRT Caenorhabditis elegans 70 Met Ser Glu Phe Leu Lys Ile Val Arg Ala Asn Lys Lys Ser Asp Arg 1 5 10 15 Lys Leu Asp Lys Thr Tyr Leu Trp Glu Ser Tyr Leu His Gln Phe Glu 20 25 30 Lys Gly Lys Thr Ser Phe Ile Pro Val Glu Ala Phe Asn Arg Asn Leu 35 40 45 Thr Val Asn Phe Asn Glu Cys Val Lys Glu Gly Val Ile Phe Glu Thr 50 55 60 Val Val His Asp Tyr Asp Lys Asn Cys Asp Ser Ile Gln Val Arg Trp 65 70 75 80 Phe Ala Arg Ile Glu Lys Val Cys Gly Tyr Arg Val Leu Ala Gln Phe 85 90 95 Ile Gly Ala Asp Thr Lys Phe Trp Leu Asn Ile Leu Ser Asp Asp Met 100 105 110 Phe Gly Leu Ala Asn Ala Ala Met Ser Asp Pro Asn Met Asp Lys Ile 115 120 125 Val Tyr Ala Pro Pro Leu Ala Ile Asn Glu Glu Tyr Gln Asn Asp Met 130 135 140 Val Asn Tyr Val Asn Asn Cys Ile Asp Gly Glu Ile Val Gly Gln Thr 145 150 155 160 Ser Leu Ser Pro Lys Phe Asp Glu Gly Lys Ala Leu Leu Ser Lys His 165 170 175 Arg Phe Lys Val Gly Gln Arg Leu Glu Leu Leu Asn Tyr Ser Asn Ser 180 185 190 Thr Glu Ile Arg Val Ala Arg Ile Gln Glu Ile Cys Gly Arg Arg Met 195 200 205 Asn Val Ser Ile Thr Lys Lys Asp Phe Pro Glu Ser Leu Pro Asp Ala 210 215 220 Asp Asp Asp Arg Gln Val Phe Ser Ser Gly Ser Gln Tyr Trp Ile Asp 225 230 235 240 Glu Gly Ser Phe Phe Ile Phe Pro Val Gly Phe Ala Ala Val Asn Gly 245 250 255 Tyr Gln Leu Asn Ala Lys Lys Glu Tyr Ile Glu His Thr Asn Lys Ile 260 265 270 Ala Gln Ala Ile Lys Asn Gly Glu Asn Pro Arg Tyr Asp Ser Asp Asp 275 280 285 Val Thr Phe Asp Gln Leu Ala Lys Asp Pro Ile Asp Pro Met Ile Trp 290 295 300 Arg Lys Val Lys Val Gly Gln Lys Phe Glu Leu Ile Asp Pro Leu Ala 305 310 315 320 Gln Gln Phe Asn Asn Leu His Val Ala Ser Ile Leu Lys Phe Cys Lys 325 330 335 Thr Glu Gly Tyr Leu Ile Val Gly Met Asp Gly Pro Asp Ala Leu Glu 340 345 350 Asp Ser Phe Pro Ile His Ile Asn Asn Thr Phe Met Phe Pro Val Gly 355 360 365 Tyr Ala Glu Lys Tyr Asn Leu Glu Leu Val Pro Pro Asp Glu Phe Lys 370 375 380 Gly Thr Phe Arg Trp Asp Glu Tyr Leu Glu Lys Glu Ser Ala Glu Thr 385 390 395 400 Leu Pro Leu Asp Leu Phe Lys Pro Met Pro Ser 405 410 71 498 PRT Caenorhabditis elegans 71 Met Ser Glu Phe Leu Lys Ile Val Arg Ala Asn Lys Lys Ser Asp Arg 1 5 10 15 Lys Leu Asp Lys Thr Tyr Leu Trp Glu Ser Tyr Leu His Gln Phe Glu 20 25 30 Lys Gly Lys Thr Ser Phe Ile Pro Val Glu Ala Phe Asn Arg Asn Leu 35 40 45 Thr Val Asn Phe Asn Glu Cys Val Lys Glu Gly Val Ile Phe Glu Thr 50 55 60 Val Val His Asp Tyr Asp Lys Asn Cys Asp Ser Ile Gln Val Arg Trp 65 70 75 80 Phe Ala Arg Ile Glu Lys Val Cys Gly Tyr Arg Val Leu Ala Gln Phe 85 90 95 Ile Gly Ala Asp Thr Lys Phe Trp Leu Asn Ile Leu Ser Asp Asp Met 100 105 110 Phe Gly Leu Ala Asn Ala Ala Met Ser Asp Pro Asn Met Asp Lys Ile 115 120 125 Val Tyr Ala Pro Pro Leu Ala Ile Asn Glu Glu Tyr Gln Asn Asp Met 130 135 140 Val Asn Tyr Val Asn Asn Cys Ile Asp Gly Glu Ile Val Gly Gln Thr 145 150 155 160 Ser Leu Ser Pro Lys Phe Asp Glu Gly Lys Ala Leu Leu Ser Lys His 165 170 175 Arg Phe Lys Val Gly Gln Arg Leu Glu Leu Leu Asn Tyr Ser Asn Ser 180 185 190 Thr Glu Ile Arg Val Ala Arg Ile Gln Glu Ile Cys Gly Arg Arg Met 195 200 205 Asn Val Ser Ile Thr Lys Lys Asp Phe Pro Glu Ser Leu Pro Asp Ala 210 215 220 Asp Asp Asp Arg Gln Val Phe Ser Ser Gly Ser Gln Tyr Trp Ile Asp 225 230 235 240 Glu Gly Ser Phe Phe Ile Phe Pro Val Gly Phe Ala Ala Val Asn Gly 245 250 255 Tyr Gln Leu Asn Ala Lys Lys Glu Tyr Ile Glu His Thr Asn Lys Ile 260 265 270 Ala Gln Ala Ile Lys Asn Gly Glu Asn Pro Arg Tyr Asp Ser Asp Asp 275 280 285 Val Thr Phe Asp Gln Leu Ala Lys Asp Pro Ile Asp Pro Met Ile Trp 290 295 300 Arg Lys Val Lys Val Gly Gln Lys Phe Glu Leu Ile Asp Pro Leu Ala 305 310 315 320 Gln Gln Phe Asn Asn Leu His Val Ala Ser Ile Leu Lys Phe Cys Lys 325 330 335 Thr Glu Gly Tyr Leu Ile Val Gly Met Asp Gly Pro Asp Ala Leu Glu 340 345 350 Asp Asn Phe Pro Ile His Ile Asn Asn Thr Phe Met Phe Pro Val Gly 355 360 365 Tyr Ala Glu Lys Tyr Asn Leu Glu Leu Val Pro Pro Asp Glu Phe Lys 370 375 380 Gly Thr Phe Arg Trp Asp Glu Tyr Leu Glu Lys Glu Ser Ala Glu Thr 385 390 395 400 Leu Pro Leu Asp Leu Phe Lys Pro Met Pro Ser Gln Glu Arg Leu Asp 405 410 415 Lys Phe Lys Val Ile Leu Ile Ser Lys Arg Val Gly Leu Arg Leu Glu 420 425 430 Ala Ala Asp Met Cys Glu Asn Gln Phe Ile Cys Pro Ala Thr Val Lys 435 440 445 Ser Val His Gly Arg Leu Ile Asn Val Asn Phe Asp Gly Trp Asp Glu 450 455 460 Glu Phe Asp Glu Leu Tyr Asp Val Asp Ser His Asp Ile Leu Pro Ile 465 470 475 480 Gly Trp Cys Glu Ala His Ser Tyr Val Leu Gln Pro Pro Lys Lys Tyr 485 490 495 Asn Tyr 72 498 PRT Caenorhabditis elegans 72 Met Ser Glu Phe Leu Lys Ile Val Arg Ala Asn Lys Lys Ser Asp Arg 1 5 10 15 Lys Leu Asp Lys Thr Tyr Leu Trp Glu Ser Tyr Leu His Gln Phe Glu 20 25 30 Lys Gly Lys Thr Ser Phe Ile Pro Val Glu Ala Phe Asn Arg Asn Leu 35 40 45 Thr Val Asn Phe Asn Glu Cys Val Lys Glu Gly Val Ile Phe Glu Thr 50 55 60 Val Val His Asp Tyr Asp Lys Asn Cys Asp Ser Ile Gln Val Arg Trp 65 70 75 80 Phe Ala Arg Ile Glu Lys Val Cys Gly Tyr Arg Val Leu Ala Gln Phe 85 90 95 Ile Gly Ala Asp Thr Lys Phe Trp Leu Asn Ile Leu Ser Asp Asp Met 100 105 110 Phe Gly Leu Ala Asn Ala Ala Met Ser Asp Pro Asn Met Asp Lys Ile 115 120 125 Val Tyr Ala Ser Pro Leu Ala Ile Asn Glu Glu Tyr Gln Asn Asp Met 130 135 140 Val Asn Tyr Val Asn Asn Cys Ile Asp Gly Glu Ile Val Gly Gln Thr 145 150 155 160 Ser Leu Ser Pro Lys Phe Asp Glu Gly Lys Ala Leu Leu Ser Lys His 165 170 175 Arg Phe Lys Val Gly Gln Arg Leu Glu Leu Leu Asn Tyr Ser Asn Ser 180 185 190 Thr Glu Ile Arg Val Ala Arg Ile Gln Glu Ile Cys Gly Arg Arg Met 195 200 205 Asn Val Ser Ile Thr Lys Lys Asp Phe Pro Glu Ser Leu Pro Asp Ala 210 215 220 Asp Asp Asp Arg Gln Val Phe Ser Ser Gly Ser Gln Tyr Trp Ile Asp 225 230 235 240 Glu Gly Ser Phe Phe Ile Phe Pro Val Gly Phe Ala Ala Val Asn Gly 245 250 255 Tyr Gln Leu Asn Ala Lys Lys Glu Tyr Ile Glu His Thr Asn Lys Ile 260 265 270 Ala Gln Ala Ile Lys Asn Gly Glu Asn Pro Arg Tyr Asp Ser Asp Asp 275 280 285 Val Thr Phe Asp Gln Leu Ala Lys Asp Pro Ile Asp Pro Met Ile Trp 290 295 300 Arg Lys Val Lys Val Gly Gln Lys Phe Glu Leu Ile Asp Pro Leu Ala 305 310 315 320 Gln Gln Phe Asn Asn Leu His Val Ala Ser Ile Leu Lys Phe Cys Lys 325 330 335 Thr Glu Gly Tyr Leu Ile Val Gly Met Asp Gly Pro Asp Ala Leu Glu 340 345 350 Asp Ser Phe Pro Ile His Ile Asn Asn Thr Phe Met Phe Pro Val Gly 355 360 365 Tyr Ala Glu Lys Tyr Asn Leu Glu Leu Val Pro Pro Asp Glu Phe Lys 370 375 380 Gly Thr Phe Arg Trp Asp Glu Tyr Leu Glu Lys Glu Ser Ala Glu Thr 385 390 395 400 Leu Pro Leu Asp Leu Phe Lys Pro Met Pro Ser Gln Glu Arg Leu Asp 405 410 415 Lys Phe Lys Val Ile Leu Ile Ser Lys Arg Val Gly Leu Arg Leu Glu 420 425 430 Ala Ala Asp Met Cys Glu Asn Gln Phe Ile Cys Pro Ala Thr Val Lys 435 440 445 Ser Val His Gly Arg Leu Ile Asn Val Asn Phe Asp Gly Trp Asp Glu 450 455 460 Glu Phe Asp Glu Leu Tyr Asp Val Asp Ser His Asp Ile Leu Pro Ile 465 470 475 480 Gly Trp Cys Glu Ala His Ser Tyr Val Leu Gln Pro Pro Lys Lys Tyr 485 490 495 Asn Tyr 73 1497 DNA Caenorhabditis elegans 73 atgtctgaat ttctgaaaat tgtcagagct aacaaaaaat cggacagaaa actcgataag 60 acctacttgt gggaatccta tttacatcag ttcgagaaag gaaaaacttc tttcattcca 120 gttgaagcat tcaatcgtaa ccttacagtt aattttaacg aatgcgtgaa ggaaggagtt 180 atcttcgaaa cagtggtcca tgattatgac aagaactgcg attcgattca agtcagatgg 240 tttgcacgaa ttgaaaaagt ttgcggatac agagttctgg ctcagtttat cggagctgac 300 acgaaatttt ggctcaatat tttatcggac gatatgtttg gtttggcaaa cgccgcaatg 360 agtgatccca atatggataa aattgtatat gctccgccgc ttgcaatcaa cgaagaatac 420 caaaatgata tggtaaatta tgtaaataat tgcattgatg gcgaaatcgt cggccaaact 480 tcgctgtctc caaaattcga tgaagggaag gctctcctaa gcaagcatcg tttcaaagtt 540 ggacaacgtc ttgaactatt aaattattcc aattctactg aaatacgcgt agcgcgaatt 600 caagaaatat gtggacgacg aatgaatgta tctatcacaa agaaagactt tcccgaatcg 660 cttccagatg cagatgacga cagacaagtc tttagctctg gatctcaata ttggatagac 720 gagggaagct tcttcatatt tcctgttgga tttgcagcag tcaatggata tcaactaaat 780 gcgaaaaagg aatatattga gcacacaaat aaaattgctc aagcaataaa aaatggagaa 840 aatccaagat atgactcaga cgacgtcaca tttgatcaat tagcaaaaga tccaattgat 900 cccatgattt ggagaaaagt taaggttgga caaaagtttg agctcatcga ccccttggct 960 cagcaattca ataacctcca cgtcgcttcg attctcaaat tttgcaaaac tgaaggatat 1020 cttattgtgg gaatggatgg tccagatgca cttgaagaca gttttcctat tcatatcaat 1080 aatacattta tgttcccagt tggttatgcg gaaaagtata atttggaact tgttccgcca 1140 gatgagttca aaggaacatt cagatgggat gaatacttgg agaaagaatc tgcagaaacc 1200 ctaccgcttg acttgttcaa gccaatgcct tcctaagaga gattagacaa atttaaggta 1260 attctgattt ccaaacgggt aggactacgc cttgaagctg ctgacatgtg tgaaaatcag 1320 tttatttgtc cagctacagt gaaatcagtt catggaagac tgataaatgt caatttcgac 1380 ggctgggatg aagaatttga tgaactgtat gatgtggact cccatgatat tctaccgata 1440 ggatggtgtg aagcgcacag ttatgttcta caacctccga aaaagtacaa ctattga 1497 74 1497 DNA Caenorhabditis elegans 74 atgtctgaat ttctgaaaat tgtcagagct aacaaaaaat cggacagaaa actcgataag 60 acctacttgt gggaatccta tttacatcag ttcgagaaag gaaaaacttc tttcattcca 120 gttgaagcat tcaatcgtaa ccttacagtt aattttaacg aatgcgtgaa ggaaggagtt 180 atcttcgaaa cagtggtcca tgattatgac aagaactgcg attcgattca agtcagatgg 240 tttgcacgaa ttgaaaaagt ttgcggatac agagttctgg ctcagtttat cggagctgac 300 acgaaatttt ggctcaatat tttatcggac gatatgtttg gtttggcaaa cgccgcaatg 360 agtgatccca atatggataa aattgtatat gctccgccgc ttgcaatcaa cgaagaatac 420 caaaatgata tggtaaatta tgtaaataat tgcattgatg gcgaaatcgt cggccaaact 480 tcgctgtctc caaaattcga tgaagggaag gctctcctaa gcaagcatcg tttcaaagtt 540 ggacaacgtc ttgaactatt aaattattcc aattctactg aaatacgcgt agcgcgaatt 600 caagaaatat gtggacgacg aatgaatgta tctatcacaa agaaagactt tcccgaatcg 660 cttccagatg cagatgacga cagacaagtc tttagctctg gatctcaata ttggatagac 720 gagggaagct tcttcatatt tcctgttgga tttgcagcag tcaatggata tcaactaaat 780 gcgaaaaagg aatatattga gcacacaaat aaaattgctc aagcaataaa aaatggagaa 840 aatccaagat atgactcaga cgacgtcaca tttgatcaat tagcaaaaga tccaattgat 900 cccatgattt ggagaaaagt taaggttgga caaaagtttg agctcatcga ccccttggct 960 cagcaattca ataacctcca cgtcgcttcg attctcaaat tttgcaaaac tgaaggatat 1020 cttattgtgg gaatggatgg tccagatgca cttgaagaca attttcctat tcatatcaat 1080 aatacattta tgttcccagt tggttatgcg gaaaagtata atttggaact tgttccgcca 1140 gatgagttca aaggaacatt cagatgggat gaatacttgg agaaagaatc tgcagaaacc 1200 ctaccgcttg acttgttcaa gccaatgcct tcccaagaga gattagacaa atttaaggta 1260 attctgattt ccaaacgggt aggactacgc cttgaagctg ctgacatgtg tgaaaatcag 1320 tttatttgtc cagctacagt gaaatcagtt catggaagac tgataaatgt caatttcgac 1380 ggctgggatg aagaatttga tgaactgtat gatgtggact cccatgatat tctaccgata 1440 ggatggtgtg aagcgcacag ttatgttcta caacctccga aaaagtacaa ctattga 1497 75 1497 DNA Caenorhabditis elegans 75 atgtctgaat ttctgaaaat tgtcagagct aacaaaaaat cggacagaaa actcgataag 60 acctacttgt gggaatccta tttacatcag ttcgagaaag gaaaaacttc tttcattcca 120 gttgaagcat tcaatcgtaa ccttacagtt aattttaacg aatgcgtgaa ggaaggagtt 180 atcttcgaaa cagtggtcca tgattatgac aagaactgcg attcgattca agtcagatgg 240 tttgcacgaa ttgaaaaagt ttgcggatac agagttctgg ctcagtttat cggagctgac 300 acgaaatttt ggctcaatat tttatcggac gatatgtttg gtttggcaaa cgccgcaatg 360 agtgatccca atatggataa aattgtatat gcttcgccgc ttgcaatcaa cgaagaatac 420 caaaatgata tggtaaatta tgtaaataat tgcattgatg gcgaaatcgt cggccaaact 480 tcgctgtctc caaaattcga tgaagggaag gctctcctaa gcaagcatcg tttcaaagtt 540 ggacaacgtc ttgaactatt aaattattcc aattctactg aaatacgcgt agcgcgaatt 600 caagaaatat gtggacgacg aatgaatgta tctatcacaa agaaagactt tcccgaatcg 660 cttccagatg cagatgacga cagacaagtc tttagctctg gatctcaata ttggatagac 720 gagggaagct tcttcatatt tcctgttgga tttgcagcag tcaatggata tcaactaaat 780 gcgaaaaagg aatatattga gcacacaaat aaaattgctc aagcaataaa aaatggagaa 840 aatccaagat atgactcaga cgacgtcaca tttgatcaat tagcaaaaga tccaattgat 900 cccatgattt ggagaaaagt taaggttgga caaaagtttg agctcatcga ccccttggct 960 cagcaattca ataacctcca cgtcgcttcg attctcaaat tttgcaaaac tgaaggatat 1020 cttattgtgg gaatggatgg tccagatgca cttgaagaca gttttcctat tcatatcaat 1080 aatacattta tgttcccagt tggttatgcg gaaaagtata atttggaact tgttccgcca 1140 gatgagttca aaggaacatt cagatgggat gaatacttgg agaaagaatc tgcagaaacc 1200 ctaccgcttg acttgttcaa gccaatgcct tcccaagaga gattagacaa atttaaggta 1260 attctgattt ccaaacgggt aggactacgc cttgaagctg ctgacatgtg tgaaaatcag 1320 tttatttgtc cagctacagt gaaatcagtt catggaagac tgataaatgt caatttcgac 1380 ggctgggatg aagaatttga tgaactgtat gatgtggact cccatgatat tctaccgata 1440 ggatggtgtg aagcgcacag ttatgttcta caacctccga aaaagtacaa ctattga 1497 76 2307 DNA Caenorhabditis elegans 76 atgctaaaat tagtcatttt gtgcttcgcg ttgttctaca atacagtcag ttcgacaaga 60 tttctgtttg gcgtcgaagt taagtgtgat tttgatgaag tgttccaatt aacagtgtcg 120 cattgggaag acgatggcaa tactttttgg gatcgcgatg aagacatcac tggacgtatg 180 actatgtttg ctcgaaagaa aatatttttc tatcaggacg gccatcatgg atttgaattt 240 ggaaagctcg agccttatgg gtggtttctg cacaattgca cgaaaaatgg aaattttcgc 300 gagtataggc acgggttgag tagcaccagt ggatccaatg ggttggagta tattgagtac 360 actgtgaatt tgacgaacgc ctgagaaata tcaaatcaaa tcgaactaac tttcaatttc 420 aataaacatt ctctctaatt acgttttaaa ccagtcttaa tttcagatgt ctgaatttct 480 gaaaattgtc agagctaaca aaaaatcgga cagaaaactc gataagacct acttgtggga 540 atcctattta catcagttcg agaaaggaaa aacttctttc attccagttg aagcattcaa 600 tcgtaacctt acagttaatt ttaacgaatg cgtgaaggaa ggagttatcg tgagttcata 660 ttgttcgtaa atcggtttta aaatacaatt tttgtagttc gaaacagtgg tccatgatta 720 tgacaagaac tgcgattcga ttcaagtcag atggtttgca cgaattgaaa aagtttgcgg 780 atacagagtt ctggctcagt ttatcggagc tgacacgaaa ttttggctca atattttatc 840 ggacgatatg tttggtttgg caaagtaagt tggacgctca gctctttcta ctattctaaa 900 taaataatgg ttctgttaca taaaattcta gagaacaatc gtattaaaac ttcgaaacat 960 ttgtataata gtaaaatttg aacatttcag cgccgcaatg agtgatccca atatggataa 1020 aattgtatat gctccgccgc ttgcaatcaa cgaagaatac caaaatgata tggtaaatta 1080 tgtaaatgta agtttgtttt tttccgaatt tatgttaata tcatctcaca acttcagaat 1140 tgcattgatg gcgaaatcgt cggccaaact tcgctgtctc caaaattcga tgaagggaag 1200 gctctcctaa gcaagcatcg tttcaaagtt ggacaacgtc ttgaactatt aaattattcc 1260 aattctactg aaatacgcgt agcgcgaatt caagaaatat gtggacgacg aatgaatgta 1320 tctatcacaa agaaagactt tcccgaatcg cttccagatg cagatgacga cagacaagtc 1380 tttagctctg gatctcaata ttggatagac gagggaagct tcttcatatt tcctgttgga 1440 tttgcagcag tcaatggata tcaactaaat gcgaaaaagg aatatattga gcacacaaat 1500 aaaattgctc aagcaataaa aaatggagaa aatccaagat atgactcaga cgacgtcaca 1560 tttgatcaat tagcaaaaga tccaattgat cccatgattt ggagaaaagt taaggttgga 1620 caaaagtttg agctcatcga ccccttggct cagcaattca ataacctcca cgtcgcttcg 1680 attctcaaat tttgcaaaac tgaaggatat cttattgtgg gaatggatgg tccagatgca 1740 cttgaagaca gttttcctat tcatatcaat aatacattta tgttcccagt tggttatgcg 1800 gaaaagtata atttggaact tgttccgcca gatgagttca aaggaacatt cagatgggat 1860 gaatacttgg agaaagaatc tgcagaaacc ctaccgcttg acttgttcaa gccaatgcct 1920 tcccaagaga gattagacaa atttaaggta attctgattt ccaaacgggt tgttttatat 1980 cgtttgagat tgtttcacta ttaatagtta ttcataattg tttcttgttt taaggtagga 2040 ctacgccttg aagctgctga catgtgtgaa aatcagttta tttgtccagc tacagtgaaa 2100 tcagttcatg gaagactgat aaatgtcaat ttcgacggct gggatgaaga atttgatgaa 2160 ctgtatgatg tggagtgagt ttatcatgac cgaacgacat tttttcaatg aaaattctat 2220 catttcagct cccatgatat tctaccgata ggatggtgtg aagcgcacag ttatgttcta 2280 caacctccga aaaagtacaa ctattga 2307 77 2307 DNA Caenorhabditis elegans 77 atgctaaaat tagtcatttt gtgcttcgcg ttgttctaca atacagtcag ttcgacaaga 60 tttctgtttg gcgtcgaagt taagtgtgat tttgatgaag tgttccaatt aacagtgtcg 120 cattgggaag acgatggcaa tactttttgg gatcgcgatg aagacatcac tggacgtatg 180 actatgtttg ctcgaaagaa aatatttttc tatcaggacg gccatcatgg atttgaattt 240 ggaaagctcg agccttatgg gtggtttctg cacaattgca cgaaaaatgg aaattttcgc 300 gagtataggc acgggttgag tagcaccagt ggatccaatg ggttggagta tattgagtac 360 actgtgaatt tgacgaacgc ctgagaaata tcaaatcaaa tcgaactaac tttcaatttc 420 aataaacatt ctctctaatt acgttttaaa ccagtcttaa tttcagatgt ctgaatttct 480 gaaaattgtc agagctaaca aaaaatcgga cagaaaactc gataagacct acttgtggga 540 atcctattta catcagttcg agaaaggaaa aacttctttc attccagttg aagcattcaa 600 tcgtaacctt acagttaatt ttaacgaatg cgtgaaggaa ggagttatcg tgagttcata 660 ttgttcgtaa atcggtttta aaatacaatt tttgtagttc gaaacagtgg tccatgatta 720 tgacaagaac tgcgattcga ttcaagtcag atggtttgca cgaattgaaa aagtttgcgg 780 atacagagtt ctggctcagt ttatcggagc tgacacgaaa ttttggctca atattttatc 840 ggacgatatg tttggtttgg caaagtaagt tggacgctca gctctttcta ctattctaaa 900 taaataatgg ttctgttaca taaaattcta gagaacaatc gtattaaaac ttcgaaacat 960 ttgtataata gtaaaatttg aacatttcag cgccgcaatg agtgatccca atatggataa 1020 aattgtatat gctccgccgc ttgcaatcaa cgaagaatac caaaatgata tggtaaatta 1080 tgtaaatgta agtttgtttt tttccgaatt tatgttaata tcatctcaca acttcagaat 1140 tgcattgatg gcgaaatcgt cggccaaact tcgctgtctc caaaattcga tgaagggaag 1200 gctctcctaa gcaagcatcg tttcaaagtt ggacaacgtc ttgaactatt aaattattcc 1260 aattctactg aaatacgcgt agcgcgaatt caagaaatat gtggacgacg aatgaatgta 1320 tctatcacaa agaaagactt tcccgaatcg cttccagatg cagatgacga cagacaagtc 1380 tttagctctg gatctcaata ttggatagac gagggaagct tcttcatatt tcctgttgga 1440 tttgcagcag tcaatggata tcaactaaat gcgaaaaagg aatatattga gcacacaaat 1500 aaaattgctc aagcaataaa aaatggagaa aatccaagat atgactcaga cgacgtcaca 1560 tttgatcaat tagcaaaaga tccaattgat cccatgattt ggagaaaagt taaggttgga 1620 caaaagtttg agctcatcga ccccttggct cagcaattca ataacctcca cgtcgcttcg 1680 attctcaaat tttgcaaaac tgaaggatat cttattgtgg gaatggatgg tccagatgca 1740 cttgaagaca gttttcctat tcatatcaat aatacattta tgttcccagt tggttatgcg 1800 gaaaagtata atttggaact tgttccgcca gatgagttca aaggaacatt cagatgggat 1860 gaatacttgg agaaagaatc tgcagaaacc ctaccgcttg acttgttcaa gccaatgcct 1920 tcccaagaga gattagacaa atttaaggta attctgattt ccaaacgggt tgttttatat 1980 cgtttgagat tgtttcacta ttaatagtta ttcataattg tttcttgttt taaggtagga 2040 ctacgccttg aagctgctga catgtgtgaa aatcagttta tttgtccagc tacagtgaaa 2100 tcagttcatg gaagactgat aaatgtcaat ttcgacggct gggatgaaga atttgatgaa 2160 ctgtatgatg tggagtgagt ttatcatgac cgaacgacat tttttcaatg aaaattctat 2220 catttcaact cccatgatat tctaccgata ggatggtgtg aagcgcacag ttatgttcta 2280 caacctccga aaaagtacaa ctattga 2307 78 2307 DNA Caenorhabditis elegans 78 atgctaaaat tagtcatttt gtgcttcgcg ttgttctaca atacagtcag ttcgacaaga 60 tttctgtttg gcgtcgaagt taagtgtgat tttgatgaag tgttccaatt aacagtgtcg 120 cattgggaag acgatggcaa tactttttgg gatcgcgatg aagacatcac tggacgtatg 180 actatgtttg ctcgaaagaa aatatttttc tatcaggacg gccatcatgg atttgaattt 240 ggaaagctcg agccttatgg gtggtttctg cacaattgca cgaaaaatgg aaattttcgc 300 gagtataggc acgggttgag tagcaccagt ggatccaatg ggttggagta tattgagtac 360 actgtgaatt tgacgaacgc ctgagaaata tcaaatcaaa tcgaactaac tttcaatttc 420 aataaacatt ctctctaatt acgttttaaa ccagtcttaa tttcagatgt ctgaatttct 480 gaaaattgtc agagctaaca aaaaatcgga cagaaaactc gataagacct acttgtggga 540 atcctattta catcagttcg agaaaggaaa aacttctttc attccagttg aagcattcaa 600 tcgtaacctt acagttaatt ttaacgaatg cgtgaaggaa ggagttatcg tgagttcata 660 ttgttcgtaa atcggtttta aaatacaatt tttgtagttc gaaacagtgg tccatgatta 720 tgacaagaac tgcgattcga ttcaagtcag atggtttgca cgaattgaaa aagtttgcgg 780 atacagagtt ctggctcagt ttatcggagc tgacacgaaa ttttggctca atattttatc 840 ggacgatatg tttggtttgg caaagtaagt tggacgctca gctctttcta ctattctaaa 900 taaataatgg ttctgttaca taaaattcta gagaacaatc gtattaaaac ttcgaaacat 960 ttgtataata gtaaaatttg aacatttcag cgccgcaatg agtgatccca atatggataa 1020 aattgtatat gctccgccgc ttgcaatcaa cgaagaatac caaaatgata tggtaaatta 1080 tgtaaatgta agtttgtttt tttccgaatt tatgttaata tcatctcaca acttcaaaat 1140 tgcattgatg gcgaaatcgt cggccaaact tcgctgtctc caaaattcga tgaagggaag 1200 gctctcctaa gcaagcatcg tttcaaagtt ggacaacgtc ttgaactatt aaattattcc 1260 aattctactg aaatacgcgt agcgcgaatt caagaaatat gtggacgacg aatgaatgta 1320 tctatcacaa agaaagactt tcccgaatcg cttccagatg cagatgacga cagacaagtc 1380 tttagctctg gatctcaata ttggatagac gagggaagct tcttcatatt tcctgttgga 1440 tttgcagcag tcaatggata tcaactaaat gcgaaaaagg aatatattga gcacacaaat 1500 aaaattgctc aagcaataaa aaatggagaa aatccaagat atgactcaga cgacgtcaca 1560 tttgatcaat tagcaaaaga tccaattgat cccatgattt ggagaaaagt taaggttgga 1620 caaaagtttg agctcatcga ccccttggct cagcaattca ataacctcca cgtcgcttcg 1680 attctcaaat tttgcaaaac tgaaggatat cttattgtgg gaatggatgg tccagatgca 1740 cttgaagaca gttttcctat tcatatcaat aatacattta tgttcccagt tggttatgcg 1800 gaaaagtata atttggaact tgttccgcca gatgagttca aaggaacatt cagatgggat 1860 gaatacttgg agaaagaatc tgcagaaacc ctaccgcttg acttgttcaa gccaatgcct 1920 tcccaagaga gattagacaa atttaaggta attctgattt ccaaacgggt tgttttatat 1980 cgtttgagat tgtttcacta ttaatagtta ttcataattg tttcttgttt taaggtagga 2040 ctacgccttg aagctgctga catgtgtgaa aatcagttta tttgtccagc tacagtgaaa 2100 tcagttcatg gaagactgat aaatgtcaat ttcgacggct gggatgaaga atttgatgaa 2160 ctgtatgatg tggagtgagt ttatcatgac cgaacgacat tttttcaatg aaaattctat 2220 catttcagct cccatgatat tctaccgata ggatggtgtg aagcgcacag ttatgttcta 2280 caacctccga aaaagtacaa ctattga 2307

Claims

What is claimed is:

1. A substantially pure nucleic acid encoding a LIN-8 polypeptide, wherein said LIN-8 polypeptide comprises at least 130 contiguous amino acids of SEQ ID NO:1 and modulates cell proliferation.

2. The nucleic acid of claim 1, wherein the amino acid sequence of said LIN-8 polypeptide comprises SEQ ID NO:1.

3. The nucleic acid of claim 1, wherein said LIN-8 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:1.

4. The nucleic acid of claim 3, wherein said LIN-8 polypeptide increases cell proliferation.

5. The nucleic acid of claim 1, wherein the polynucleotide sequence of said nucleic acid comprises SEQ ID NO:2.

6. The nucleic acid of claim 1, wherein the polynucleotide sequence of said nucleic acid comprises at least 400 contiguous nucleotides of SEQ ID NO:2.

7. The nucleic acid of claim 6, wherein said polynucleotide sequence of said nucleic acid comprises a mutant lin-8 nucleic acid sequence.

8. The nucleic acid of claim 7, wherein said polynucleotide sequence comprises any one of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.

9. A mutant lin-8 nucleic acid having a polynucleotide sequence comprising SEQ ID NO:20.

10. A mutant lin-8 nucleic acid having a polynucleotide sequence comprising SEQ ID NO:22.

11. A polypeptide having an amino acid sequence identical to any one of SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:47.

12. A substantially pure nucleic acid encoding a LIN-56 polypeptide, wherein said LIN-56 polypeptide comprises at least 110 contiguous amino acids of SEQ ID NO:3 and modulates cell proliferation.

13. The nucleic acid of claim 12, wherein the amino acid sequence of said LIN-56 polypeptide comprises SEQ ID NO:3.

14. The nucleic acid of claim 12, wherein said LIN-56 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:3.

15. The nucleic acid of claim 14, wherein said LIN-56 polypeptide increases cell proliferation.

16. The nucleic acid of claim 12, wherein the polynucleotide sequence of said nucleic acid comprises SEQ ID NO:4.

17. The nucleic acid of claim 12, wherein the polynucleotide sequence of said nucleic acid comprises at least 400 contiguous nucleotides of SEQ ID NO:4.

18. The nucleic acid of claim 17, wherein said polynucleotide sequence of said nucleic acid comprises a mutant lin-56 nucleic acid sequence.

19. The nucleic acid of claim 18, wherein said polynucleotide sequence comprises SEQ ID NO:48.

20. A substantially pure nucleic acid encoding a LIN-61 polypeptide, wherein said LIN-61 polypeptide comprises at least 130 contiguous amino acids of SEQ ID NO:5 and modulates cell proliferation.

21. The nucleic acid of claim 20, wherein the amino acid sequence of said LIN-61 polypeptide comprises SEQ ID NO:5.

22. The nucleic acid of claim 20, wherein said LIN-61 polypeptide has an amino acid alteration relative to the sequence of SEQ ID NO:5.

23. The nucleic acid of claim 22, wherein said LIN-61 polypeptide increases cell proliferation.

24. The nucleic acid of claim 20, wherein the polynucleotide sequence of said nucleic acid comprises SEQ ID NO:6.

25. The nucleic acid of claim 20, wherein the polynucleotide sequence of said nucleic acid comprises at least 400 contiguous nucleotides of SEQ ID NO:6.

26. The nucleic acid of claim 25, wherein said polynucleotide sequence of said nucleic acid comprises a mutant lin-61 nucleic acid sequence.

27. The nucleic acid of claim 26, wherein said polynucleotide sequence comprises any one of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:78.

28. A polypeptide having an amino acid sequence identical to any one of SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.

29. A vector comprising a nucleic acid having a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

30. A transgenic cell comprising a nucleic acid sequence encoding a lin-8, a lin-56, or a lin-61 polypeptide, wherein said nucleic acid sequence is located in the genome of said cell in a position in which it does not naturally occur.

31. The method of claim 30, wherein said nucleic acid sequence is operably linked to a heterologous promoter.

32. A purified antibody which specifically binds to a LIN-8 polypeptide.

33. A purified antibody which specifically binds to a LIN-56 polypeptide.

34. A purified antibody which specifically binds to a LIN-61 polypeptide.

35. A method of modulating proliferation of a cell, said method comprising administering to said cell a proliferation-modulating amount of a polypeptide having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

36. The method of claim 35, wherein said cell is in a mammal.

37. The method of claim 36, wherein said mammal is a human.

38. A method of modulating proliferation of a cell, said method comprising administering to said cell a proliferation-modulating amount of a nucleic acid sequence encoding a polypeptide having the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

39. The method of claim 38, wherein said nucleic acid sequence is contained in a vector.

40. A method of identifying a compound that modulates cell proliferation, said method comprising:

(a) providing a cell expressing a nucleic acid operably linked to a lin-8, lin-56, or lin-61 promoter;

(b) contacting said cell with a candidate compound; and

(c) measuring the expression of said nucleic acid, wherein an alteration in the level of expression of said nucleic acid indicates the presence of a compound that modulates cell proliferation.

41. The method of claim 40, wherein said nucleic acid is selected from the group consisting of lin-8, lin-56, and lin-61.

42. The method of claim 40, wherein said nucleic acid is a reporter gene.

43. The method of claim 40, wherein step (c) comprises measuring the expression of the protein encoded by said nucleic acid.

44. The method of claim 43, wherein said protein is contacted with an antibody that specifically binds to a LIN-8, LIN-56, or LIN-61 polypeptide.

45. The method of claim 40, wherein step (c) comprises measuring the mRNA level of said nucleic acid.

46. A method of identifying a candidate compound that binds to a LIN-8, LIN-56, or LIN-61 polypeptide, said method comprising:

(a) providing said polypeptide;

(b) contacting said polypeptide with a candidate compound; and

(c) measuring the binding of said candidate compound to said polypeptide, said binding indicating the presence of a candidate compound that binds a LIN-8, LIN-56, or LIN-61 polypeptide.

47. The method of claim 46, wherein said candidate compound is a polypeptide.

48. A method of diagnosing an animal for the presence of a cell proliferation disease, or an increased likelihood of developing a cell proliferation disease, said method comprising, determining whether a nucleic acid sample obtained from said animal comprises a mutant lin-8, lin-56, or lin-61 nucleic acid, wherein the presence of said mutant lin-8, lin-56, or lin-61 nucleic acid indicates that said animal has a cell proliferation disease, or is at an increased likelihood of developing a cell proliferation disease.

49. The method of claim 48, wherein said mutant lin-8 nucleic acid is selected from the group consisting of lin-8(n2738), lin-8(n2731), lin-8(n3606), lin-8(n3595), lin-8(n2739), lin-8(n3586), lin-8(n3588), lin-8(n-111), lin-8(n2741), lin-8(n3585) 8(n3646), lin-8(n2376), lin-8(n2378), lin-8(n2403), lin-8(n2724), lin-8(n3585), lin-8(n3591), lin-8(n3609), and lin-8(n3581).

50. The method of claim 48, wherein said mutant lin-56 nucleic acid is a lin-56(n3355) or lin-56(n2728) nucleic acid.

51. The method of claim 48, wherein said mutant lin-61 nucleic acid is selected from the group consisting of lin-61(n3446), lin-61(n3447), lin-61(n3624), and lin-61(n3635).

52. The method of claim 48, wherein said cell proliferation disease is cancer.

53. A method of diagnosing an animal for the presence of a cell proliferation disease, or an increased likelihood of developing a cell proliferation disease, said method comprising measuring lin-8, lin-56, or lin-61 nucleic acid expression in a sample obtained from said animal, wherein an alteration in said expression, relative to a sample obtained from an unaffected animal, indicates that said animal has a cell proliferation disease, or an increased likelihood of developing a cell proliferation disease.

54. The method of claim 53, wherein said nucleic acid expression is measured by measuring the amount of said LIN-8, LIN-56, or LIN-61 polypeptide in said sample.

55. The method of claim 54, wherein said amount of said LIN-8, LIN-56, or LIN-61 polypeptide is measured using an antibody that specifically binds to a LIN-8, LIN-56, or LIN-61 polypeptide.

56. The method of claim 53, wherein said nucleic acid expression is measured by measuring the amount of lin-8, lin-56, or lin-61 mRNA in said sample.

57. The method of claim 53, wherein said animal is a mammal.

58. The method of claim 57, wherein said mammal is a human.

59. A method of identifying a nucleic acid that modulates cell proliferation, said method comprising:

(a) expressing in a cell (i) a first nucleic acid operably linked to a first promoter, wherein said first promoter is selected from the group consisting of the lin-8, lin-56, and lin-61 promoter; and (ii) a second nucleic acid operably linked to a second promoter; and

(b) measuring the expression of said first nucleic acid, wherein a modulation in said expression of said first nucleic acid in the presence of said second nucleic acid, indicates that said second nucleic acid modulates cell proliferation.

60. The method of claim 59, wherein said first nucleic acid is selected from the group consisting of a lin-8, lin-56, and lin-61 nucleic acid.